RESEARCH ARTICLE

Injury affects coelomic fluid proteome of the common starfish,Asterias rubensSergey V. Shabelnikov1,*, Danila E. Bobkov2, Natalia S. Sharlaimova2 and Olga A. Petukhova2

ABSTRACTEchinoderms, possessing outstanding regenerative capabilities,provide a unique model system for the study of response to injury.However, little is known about the proteomic composition of coelomicfluid, an important biofluid circulating throughout the animal’s bodyand reflecting the overall biological status of the organism. In thisstudy, we used LC-MALDI tandemmass spectrometry to characterizethe proteome of the cell-free coelomic fluid of the starfish Asteriasrubens and to follow the changes occurring in response to puncturewound and blood loss. In total, 91 proteins were identified, of which61 were extracellular soluble and 16 were bound to the plasmamembrane. The most represented functional terms were ‘patternrecognition receptor activity’ and ‘peptidase inhibitor activity’. A seriesof candidate proteins involved in early response to injury wasrevealed. Ependymin, β-microseminoprotein, serum amyloid A andavidin-like proteins, which are known to be involved in intestinalregeneration in the sea cucumber, were also identified as injury-responsive proteins. Our results expand the list of proteins potentiallyinvolved in defense and regeneration in echinoderms anddemonstrate dramatic effects of injury on the coelomic fluid proteome.

KEY WORDS: Echinoderms, Biofluid, Injury response, Wound,HPLC, LC-MALDI

INTRODUCTIONExtracellular body fluids, such as blood plasma in vertebratesand hemolymph and coelomic fluid (CF) in invertebrates, serve as atransport system for metabolites, nutrients, signaling and defensemolecules in virtually all animals. Extracellular body fluids containsoluble factors that are constitutively secreted as well as those thatare released under specific physiological conditions from differentsources throughout the entire body, providing an opportunity todevelop an overall profile of the biological status of the organism.Various soluble factors are directly or indirectly involved indifferent physiological processes, such as wound healing,inflammation, tissue remodeling and cell migration. These solublefactors will leak into the extracellular body fluids, serving asbiomarkers of their corresponding processes.Echinoderms have a well-developed perivisceral coelom

(Lawrence, 1987) in which CF circulates through the whole body,bathing internal tissues and organs. The volume of CF constitutes asignificant fraction of the whole body mass, reaching 20% in starfish

and exceeding 40% in sea urchins (Giese, 1966). Cellular elementsfound in CF at concentrations of 105–106 cells ml−1 are collectivelyreferred to as coelomocytes (Chia and Xing, 1996). Theirmorphology in starfish is well described (Kanungo, 1984;Sharlaimova et al., 2014). Coelomocytes are not only mediators ofinnate immune responses (Smith et al., 2010), but are also activelyinvolved in the early repair phase of starfish arm regeneration (BenKhadra et al., 2017; Ferrario et al., 2018), providing wound closureand ‘hemostatic’ activity. The number of circulating coelomocytes instarfish rapidly increases in the first hours after arm tip amputation(Pinsino et al., 2007) or an immune challenge (Coteur et al., 2002;Holm et al., 2008), indicating the importance of cellular componentsin the early post-traumatic period.

Previously, two injury models have been established in Asteriasrubens (Kozlova et al., 2006; Sharlaimova et al., 2014) to study thedynamics of the CF cell population during the early regenerationperiod: puncture wound (PW) and blood loss (BL). The PWmodel ischaracterized bymaking a single puncture into the body wall, leadingto slight bleeding, whereas BL involves arm tip amputation,excessive bleeding and artificial washing of the coelomic cavity,effectively reducing the CF cell pool by up to 90% (Sharlaimovaet al., 2014). These injuries are clearly different in their consequences:PW induces a rapid and transient increase in the number of circulatingcoelomocytes in the few hours after injury with its cellularcomposition remaining unchanged (Gorshkov et al., 2009; Kozlovaet al., 2006), whereas BL is followed by changes in the cellularcompositions of both CF and coelomic epithelium, upregulation ofprotein synthesis in coelomocytes, and migration of small, poorlydifferentiated cells from the coelomic epithelium into the CF(Kozlova et al., 2006; Sharlaimova et al., 2014). Involvement ofsmall, poorly differentiated cells in the renewal of CF cell populationsafter BL injury has been proposed (Sharlaimova et al., 2014).

Echinoderms, having an outstanding capability to regenerate bodyparts and even complete individuals from a fragment following self-induced or traumatic amputation processes, represent valuable modelsin regeneration research (Ben Khadra et al., 2017; Candia-Carnevaliet al., 2009; Dolmatov, 1999). Recent high-throughput studies ofregeneration in sea cucumbers (Dolmatov et al., 2018; Zhang et al.,2017) have enabled discovery of new players involved in this process;unfortunately, nothing is known about the protein composition of theCF in these animals. Previous proteomic studies of the echinodermCFhave generally focused on immune-challenge- and age-relatedchanges in sea urchins (Bodnar, 2013; Dheilly et al., 2011; Dheillyet al., 2012; Dheilly et al., 2013). These studies analyzed whole CFwithout thorough removal of coelomocytes, which apparentlyhampered identification of scarce extracellular components in favorof highly abundant intracellular proteins. Although several proteomicstudies have been performed on starfish analyzing mucous secretions(Hennebert et al., 2015), nervous system regeneration (Franco et al.,2014) and coelomocytes (Franco et al., 2011), none have addressedthe protein composition of CF or its injury-related changes.Received 19 December 2018; Accepted 11 February 2019

1Laboratory of Regulation of Gene Expression, Institute of Cytology, RussianAcademy of Sciences, 194064 St Petersburg, Russia. 2Department of Cell Cultures,Institute of Cytology, Russian Academy of Sciences, 194064 St Petersburg, Russia.

*Author for correspondence (sergey.v.shabelnikov@gmail.com)

S.V.S., 0000-0002-5693-5310

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Here, we present a proteomic analysis of cell-free CF samplesfrom A. rubens. This echinoderm provides a suitable modelfor proteomic analysis of the CF for the following reasons. First,regenerating specimens of A. rubens are frequently found in thewild, suggesting the existence of well-developed healingmechanisms. Second, A. rubens has well-developed perivisceralcoelom in terms of both volume and size, permitting multiplerounds of collection of CF. Third, there are three A. rubenstranscriptomic datasets available (Hennebert et al., 2015;Reich et al., 2015; Semmens et al., 2016), enabling the creationof a species-specific protein database. Forth, A. rubens is acommon and easily obtained species of starfish in the White Seain Russia.Essentially, the main question we ask is: if an animal with high

regenerative capacity and well-developed coelom is subjected toinjury, can we identify the soluble proteins involved and are theserelated to regeneration? To address this question, we examined CFin the course of the early repair phase in two well-established injurymodels. The overall aims of this study were to (1) provide anoverview of the starfish cell-free CF proteome and (2) describe theimpact of injury on CF composition and reveal the injury-relatedsoluble factors involved.

MATERIALS AND METHODSEthics statementSteps were taken to ensure that animals did not suffer unnecessarilyduring any stage of the experiments. The animals were returnedalive to their natural environment after experimentation.

Animals and experimental injuryExperiments were performed at the Biological Station of theZoological Institute, Russian Academy of Sciences, on CapeKartesh (Kandalaksha Bay, White Sea) in September 2016. Adultindividuals of the common starfish, Asterias rubens Linnaeus 1758,were collected off Fettakh Island (66°20′05″ N, E33°39′07″ E).Asterias rubens is not a protected or endangered species. Animalswere kept in cages at a depth of 5–6 m throughout the experimentalperiod. We also included a 3-day acclimation and starvation periodprior to experimentation in order to minimize possible perturbationsof CF caused by the animal feeding and capture procedure. Thestarfish used had a radius ranging from 5 to 10 cm,measured from thelargest arm to the center of the oral disc. Two types of experimentaltraumatic treatment were used: puncture wound (PW) and blood loss(BL). The experimental workflow is shown in Fig. 1. The PW (n=12individuals, mean radius=8.3 cm) was inflicted by puncturing theaboral surface of the arm with a scalpel. The puncture resulted in anincision of approximately 10 mm and minor bleeding. The BL injury(n=8 individuals, mean radius=5.4 cm)was performed as follows: thearm tip was cut off, and the CF was drained off as completely aspossible. Then, the coelomic cavity was washed with four 4 mlinjections of 0.22 µm-filtered seawater into each arm tip, and theinjected seawater was drained off to remove residual CF andcoelomocytes. The injured animals were returned to their cages.

Experimental designTwo pilot studies were performed before analysis of injuryresponses. Firstly, individual samples of CF from untreated,

SPE protein extraction and digestion HPLC analysis(MiLiChrom A-02 and Jupiter C5)

Pooled 12 or 8 samplesSingle replicate

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Fig. 1. Schematic overview of the experimental workflow. A repeated-measures design was used to reduce the effects of biological variance amongindividuals; therefore, control and time-point samples were taken from the same individual. Two traumatic treatments [puncture wound (PW) and blood loss (BL)]and five experimental groups (CPW, PW6h, CBL, BL6h and BL72h), each with one pooled biological replicate (12 individuals in PW, and eight individuals in BL)were analyzed, yielding a list of identified coelomic fluid (CF) proteins.

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recently captured starfish (n=23, mean radius=8.3±1.1 cm) wereanalyzed by reverse-phase HPLC to assess individual variability.Descriptive statistics are presented as means±s.d. Secondly, theeffect of repeated sampling of CF on the protein profile of CF wasexamined by HPLC (n=5, mean radius=6.4±0.5 cm). Groupdifferences across time in peak areas (proportions from total peakarea) were evaluated using repeated-measures ANOVA, withsampling (before sampling and 6 h after sampling) as the withinsubjects factor. Arcsine square root transformation was used tostabilize variance and normalize proportional data.To follow the changes occurring in response to PW and BL, we

utilized a repeated-measures design to reduce the effect ofbiological variance among individuals. Repeated measures wererealized as repeated sampling of CF from the same animals atdifferent time points. The experimental workflow is shown in Fig. 1.A total of 12 animals were taken for PW, and eight animals for BL.Two time points were taken for PW (before injury and 6 h afterinjury), and three time points for BL injury (before injury, and 6 and72 h after injury). Samples collected before injuries were used ascontrols for protein abundance changes caused by injuries. Samplesfrom the same injury type and time point were pooled together toprovide sufficient protein material. Four independent LC-MALDI-TOF/TOF acquisitions and one HPLC analysis of each pooledsample were performed. Therefore, two traumatic treatment (PWand BL) and five experimental groups (CPW, PW6h, CBL, BL6hand BL72h) each with one pooled biological replicate (pool of 12samples in PW, and pool of eight samples in BL) were analyzed.Thus, our inference for each experimental group is based oncomparisons of single pooled biological replicates and fourLC-MALDI technical replicates complemented by HPLC analysisof undigested CF sample.

Sample collectionThe samples of CF were collected 6 h after puncture wound (PW6h)and 6 and 72 h after blood loss (BL6h and BL72h, respectively).Samples collected just before injury served as controls (CPW andCBL). The CFwas obtained from the coelomic cavity by puncturingthe aboral epidermis at the arm tip with a 21 G double-ended needle(Fig. 2A) and collecting 1.5 ml of the CF by gravity flow into amicrotube containing 45 μl of 0.5 mol l−1 EDTA, pH 7.5. The armtip opposite to the wounded one was used for sampling. Two low-speed centrifugations were performed to minimize damage to thefragile cells from excessive centrifugation and minimize potentialcontamination of the CF with non-secretory cellular proteins.Firstly, samples were centrifuged at 120 g for 10 min in a bucketrotor. Then, the supernatant was collected and subjected tocentrifugation at 430 g for 10 min in a bucket rotor, followed byfiltration with Spin-X centrifuge tube filters (0.45 μm, celluloseacetate membrane, Corning Costar) at 732 g for 5 min in aMiniSpinplus centrifuge (Eppendorf, Hamburg, Germany). The flow-throughaliquots of 500 μl from independent samples of each experimentalgroup were pooled (n=12 for PW, n=8 for BL), resulting in 6 and4 ml of cell-free CF, respectively. The samples were snap-frozen inliquid nitrogen and stored at −80°C pending analysis. Each pooledsample was analyzed by HPLC.

HPLC analysis of CFAnalysis of pooled CF samples was performed on a microboreHPLC system (MiLiChrom A-02, EcoNova, Novosibirsk, Russia).A sample volume of 100 μl was loaded onto a Jupiter C5 reversed-phase column (2×100 mm, 5 μm, 300 Å, Phenomenex, Torrance,CA, USA) and separated using a linear gradient of 15–80% B over22 min at a flow rate of 200 μl min−1. The mobile phases used were

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Fig. 2. Evaluation of the coelomic fluid sampling procedure. (A) Sampling of the coelomic fluid from the arm tip of the starfish Asterias rubens.(B) Representative HPLC profiles of individual samples from untreated, recently captured starfish show individual variability. (C) Variability of the eight mostabundant protein peaks for 23 samples. The means (squares), medians (lines), 25th to 75th percentiles (boxes), 10th to 90th percentiles (whiskers) and extremevalues (asterisks) are shown. (D) HPLC profiles of individual samples obtained from five normal starfish (C1–C5) by two repeated samplings with a 6 hinterval (first sampling: gray line; second sampling: red line). (E) Arcsine square root transformed proportions of total peak area for seven proteins (first sampling:gray line; second sampling: red line). Data points are the means (squares) with 95% confidence intervals (whiskers). Peaks are marked according to Fig. 8.

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A, 0.125% (v/v) trifluoroacetic acid (TFA) in water, and B, 0.125%(v/v) TFA in acetonitrile. The column was maintained at 45°C.Absorbance was monitored at 206, 250 and 280 nm, with aUV detector slit width of 5 nm. Total protein concentration wasestimated from the total peak area using A205 nm=31 ml mg−1 cm−1 (Scopes, 1974). Major peaks were collectedand in-solution digested with trypsin/Lys-C mix and subjected toMALDI TOF/TOF analysis.

Cell countingThe coelomocyte count data were obtained for each individualsample by counting formalin-fixed cells using a hemocytometer.The data were compared by non-parametric Wilcoxon matchedpairs test. Data are presented as means±s.d.

Protein extraction and digestionPooled CF samples were subjected to solid-phase extraction usingOasis HLB CC (Waters, Milford, MA, USA) and Strata C18-T100 mg (Phenomenex) solid-phase extraction tubes sequentiallyconnected and equilibrated with 10% v/v acetonitrile and 0.1% v/vTFA. Unbound compounds were removed by washing with 5 ml ofequilibration buffer, and proteins were eluted with 1.5 ml of 90% v/vacetonitrile and 0.1% v/v TFA independently from both tubes. Eluteswere mixed and dried with a rotor vacuum evaporator. The sampleswere dissolved in 100 μl of aqueous 5% v/v sodium deoxycholate(DOC) and incubated at 95°C for 5 min. In-solution digestion wasperformed according to the DOC protocol (Proc et al., 2010).Denatured samples were subsequently reduced with 100 μl of20 mmol l−1 dithiothreitol in 50 mmol l−1 ammonium bicarbonatefor 30 min at 60°C and alkylated with 100 μl of 50 mmol l−1

iodoacetamide in 50 mmol l−1 ammonium bicarbonate in the dark for30 min at room temperature. The samples were diluted by theaddition of 300 μl of 50 mmol l−1 ammonium bicarbonate, anddigested for 18 h at 37°Cwith 0.5 μg of trypsin/Lys-Cmix (Promega,Madison, WI, USA). The digestion was stopped and the DOC wasprecipitated by acidifying the sample with 50 μl of 1% TFA andvortexing. Digested samples were centrifuged at 14,000 g for 10 minwith a MiniSpin plus centrifuge (Eppendorf) to pellet the DOC. Alldigests were then desalted and concentrated with solid-phaseextraction tips packed with 20 mg of Strata C18-T sorbent(Phenomenex). The samples were eluted with 600 μl of 90% v/vacetonitrile and 0.1% v/v TFA and dried with a rotor vacuumevaporator. After rehydration with 0.1% TFA, the samples werefiltered and divided into four 50 μl aliquots that were used as technicalreplicates in the LC-MALDI-MS/MS analyses.

Peptide fractionationPeptides were separated with a Jupiter Proteo C12 reversed-phasecolumn (1×50 mm, 4 μm, 90 Å, Phenomenex) on a microboreHPLC system (MiLiChrom A-02, EcoNova). A sample volume of50 μl was injected and separated using a linear gradient of 10–35%B over 54 min followed by 35–90% B for 6 min at a flow rate of50 μl min−1. The mobile phases used were A, 0.125% (v/v) TFA inwater, and B, 0.125% (v/v) TFA in acetonitrile. The column wasmaintained at 45°C. The effluent from the HPLC column wasmixed with α-cyano-4-hydroxycinnamic acid (CHCA) matrix(12 mg ml−1 in 95% acetonitrile) at a flow rate of 15 μl min−1 via amicro tee. An in-house constructed Arduino-powered micro-fraction collector was used to deposit a total of 912 fractions of0.5 μl in a 24×38 array on an LC-MALDI plate (SCIEX,Darmstadt, Germany). The column was washed with a saw-toothgradient (15–80% for 4 min followed by 80–15% for 2 min,

repeated eight times) and equilibrated to 10% B for 10 min beforesubsequent injections.

MALDI-TOF/TOF mass spectrometryThe fractionated samples were analyzed with a TOF/TOF 5800System (SCIEX) instrument operated in the positive ion mode. TheMALDI stage was set to continuous motion mode. MS data wereacquired at 2400 laser intensity with 1000 laser shots/spectrum (200laser shots/sub-spectrum) and MS/MS data were acquired at 3300laser intensity with a DynamicExit algorithm and a high spectralquality threshold or a maximum of 1000 laser shots/spectrum (250laser shots/sub-spectrum). Up to 35 top precursors with signal tonoise ratio >30 in the mass range 850–4000 Da were selected fromeach spot for MS/MS analysis.

Protein identificationFour independent LC-MALDI-TOF/TOF acquisitions of eachpooled sample were performed and processed together in one runwith the Protein Pilot 4.0 software (SCIEX). The Paragon algorithm4.0 was used in thorough mode with biological modifications andsubstitutions enabled. Carbamidomethyl cysteine was set as a fixedmodification. The subject of Paragon searches was a pooled proteindatabase comprising protein-coding open reading frames (ORFs)predicted from three A. rubens transcriptome shotgun assemblydatasets: ovary (Reich et al., 2015), tube foot (Hennebert et al.,2015) and radial nerve (Semmens et al., 2016). Each transcriptomeshotgun assembly dataset was subjected to two iterativereassemblies with iAssembler (Zheng et al., 2011). Then, ORFswere predicted with TransDecoder-v5.0.2 (Haas et al., 2013) at aminimum ORF length of 70 amino acids and using the homologyoption. Searches with HMMSCAN (Eddy, 2011) against Pfam-Aversion 31.0, and with BLASTP against a set of Echinodermatasequences downloaded from UniProtKB on 12 December 2017were used for the homology option. Redundant sequences were thenremoved using CD-HIT (Li and Godzik, 2006) at a 97% identitythreshold, local alignment and 80% alignment coverage for shortersequences (-c 0.97, -G 0 and -aS 0.8 options), producing 24,796ORFs. These were then enriched with a set of 947 non-redundantsecretory and single-pass transmembrane protein-coding sequencespredicted from the ovary transcriptome shotgun assembly ofAsterias forbesi as described below. The resulting set was filteredwith CD-HIT, producing a final database of 25,725 protein-codingsequences. The database also incorporated a list of commoncontaminants. False discovery rate (FDR) analysis was done byanalysis of reversed sequences using the embedded PSEP tool. Themass spectrometry proteomics data and search database have beendeposited at the ProteomeXchange Consortium via the PRIDEpartner repository with the dataset identifier PXD010228.

Proteomics data filtering and determination of differentialabundanceWe estimated fractions of secretory soluble and single-passtransmembrane proteins in our database as ∼0.13 and ∼0.11,respectively. Therefore, considering the low probability ofidentification of extracellular proteins by chance and assumingthat CF predominantly contains secreted proteins and, to a lesserextent, shed membrane protein ectodomains, we decided to acceptidentifications of proteins having predicted signal sequences withone high-scoring peptide spectrum match (PSM; ProteinPilotpeptide confidence of 99%) and a ProteinPilot unused score >1.3,but proteins without signal sequences were retained if they wereidentified with ≥2 PSM and a ProteinPilot unused score >2. The

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use of goodness-of-fit tests for differential protein abundancedetermination in proteomic experiments with single replicates hasbeen validated (Zhang et al., 2006). The log-likelihood ratio test(G-test) (Zar, 2014) comparing frequencies in a sample withfrequencies hypothesized in the sampled population was used. Thenull hypothesis tested was that there is no significant differencebetween observed and expected spectral counts. Observed spectralcounts were spectral counts observed after treatment, whereasexpected spectral counts were derived from control spectral countsby scaling to the total spectral count of a treatment condition,assuming that observed differences are result of change of totalprotein amount. William’s correction was used for adjustment of theG-statistic. Then, the McNemar test of symmetry for paired sampleswas performed for post hoc analysis (Zar, 2014). The nullhypothesis tested was that the ratio of spectral counts forparticular protein before and after treatment is 1:1. The McNemarstatistic in the form of a Pearson chi-square with Yates correction is:

x2c ¼ðjse � scj � 1Þ2

se þ sc; ð1Þ

where se and sc are the numbers of PSMs in experimental andcontrol samples, respectively. The number of PSMs includedpeptides reported by ProteinPilot software at a ≥95% confidencethreshold. Therefore, to classify proteins as differentially abundant,the ratio of spectral counts should significantly (P<0.05) deviatefrom 1:1. All relevant data are provided in Table S2. We note thatabove-mentioned criterion was applied only to proteins identifiedboth in control and experimental samples, whereas all proteinsidentified exclusively in experimental samples were considered asdifferentially abundant for this particular treatment. A proteinabundance index (PAI) (Ishihama et al., 2005) was used as ameasure of protein abundance for graphical representation bymeansof a heat map. The PAI was defined as:

PAI ¼ Nobserved

Nobservable; ð2Þ

where Nobserved is the number of PSMs and Nobservable is the numberof expected peptides for the protein. If non-tryptic cleavages weredetected, resulting in overrepresentation of the number of observedpeptides, count data were corrected by addition of the number ofobserved non-tryptic cleavage sites to the number of expectedpeptides in Eqn 2. The list of expected peptides for the protein wasobtained by in silico digestion with PeptideMass (Wilkins et al.,1997) with no missed cleavages, after removal of the signal peptide,and filtered by both a mass range of 850–4000 Da and ahydrophobicity index range of 4.9–32.2 a.u. calculated withSSRCalc (Krokhin, 2006).

Bioinformatic analysis of proteomic dataThe number of secretory soluble and single-pass transmembraneproteins in our database was estimated using Phobius (Käll et al.,2004). A fraction of sequences with signal peptide only, andsequences with one transmembrane region starting ≤30 amino acidresidues from the N-terminus was accepted as a reasonableestimation of secretory soluble proteins. The fraction of single-pass transmembrane proteins was estimated as the sum of sequenceshaving a signal peptide and one transmembrane region, sequenceswith one transmembrane region starting >30 amino acid residuesfrom the N-terminus, and sequences containing two transmembraneregions starting ≤30 amino acid residues from the N-terminus.

Similarity searches in the UniProtKB database were performedusing BLASTP implemented in Blast2GO tool (Conesa et al., 2005)at an E-value threshold of 10−5. TBLASTN searches wereperformed against the TSA and EST databases available at theNCBI server and restricted to Echinodermata. Sequences withundetectable similarity were submitted to HHblits at the MPIBioinformatics Toolkit server (Zimmermann et al., 2018) underdefault settings, and the resulting multiple sequence alignmentswith E-values lower than 10−5 were entered into the HHpred serverto search against the Pfam-A version 31.0 and SMART version 6.0databases. Domain organization was analyzed with a SMART webtool (Letunic and Bork, 2018). Protein domain architectures witheither different domain counts or different order were computed asindependent domain combinations, even if they had the samedomain types. Functional annotation was based on sequencesimilarity, conserved domain searches and similarity of domainorganization with well-characterized proteins. If no functional termswere assigned via the BLAST search, putative terms were suggestedbased on conserved domain gene ontology (GO) terms andfunctions of proteins with similar domain organization.Glycosylphosphatidylinositol (GPI) anchoring was predicted withPredGPI (Pierleoni et al., 2008). Signal peptides were predictedwith SignalP 4.0 (Petersen et al., 2011), and transmembrane regionswere identified as a consensus of Phobius and TMHMM (Kroghet al., 2001) predictors.

RESULTSComposition of the CF varies among individualsTo assess the CF composition variability, we examined individualsamples from 23 untreated, recently captured starfish, with the useof reversed-phase HPLC. Substantial inter-individual differenceswere observed (Fig. 2B). The number of detected peaks varied from11 to 19, with mean value of 15 peaks. Total peak area varied from11.8 to 145.1 a.u. µl, with mean value of 46.6±29.7 a.u. µl, and acoefficient of variation (CV) of 63.9%. Total protein concentrationwas roughly estimated as 15.0±9.6 µg ml−1. No correlation betweentotal peak area and starfish size was observed. We then estimatedvariability by the eight most abundant peaks occupying 91.9±6.3%of total peak area. All peaks showed a right-tailed distribution withhigh variance and a CV ranging from 55% to 204% (Fig. 2C;Table S1). An interesting finding was a strong, positive correlationbetween the WAPL and KUTZ peaks (R=0.82, P=0.0001).

The observed inter-individual variability motivated us to use arepeated-measures experimental design as an effective way toexclude the effects of individual differences. Then, we examined theeffect of repeated sampling of CF. No significant differencesbetween HPLC profiles of CF samples collected 6 h apart wereobserved (Fig. 2D,E). Thus, starfish appear to be insensitive to theCF sampling method used.

The proteome of cell-free CFWithin the four experimental groups, we identified in total 119proteins with a local FDR of 5%. The identification list was thenfiltered against a stringent threshold, leading to a final list of 91accepted protein identifications (Table S2). Of these, 23 sequenceswere putative secretory proteins identified with only one high-scoring PSM (Figs S1–S23), retained so as not to exclude potentialtargets that may have been near the limit of detection. The positionof the signal peptide cleavage site was confirmed for 20 proteins byidentification of a corresponding peptide with an N-terminal non-tryptic cleavage site (Table S2). Processing of CRH-type peptideand progranulin into mature forms was confirmed by detection in

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the CF of fragments originating only from the predicted maturepeptides. In spite of our database containing a partial codingsequence for progranulin, we detected four mature peptidescorresponding to human granulins 3, 4, 5 and 6 (Fig. S24).

Functional annotation and domain organizationAs expected, computational analysis based on SignalP, TMHMM,Phobius and PredGPI predictions showed that 77 of 91 proteinsidentified were predicted to be extracellular or bound to the plasmamembrane. The proteins were grouped into four cellular locationcategories (Fig. 3A). Putative functions of identified proteins wereassigned by searching for homologous sequences in the UniProt KBdatabase using the Blast2GO tool. A relatively high number ofsequences (59) showed similarity (at an E-value threshold of 10–5)with annotated proteins, whereas 16 sequences showed homologyto uncharacterized Strongylocentrotus purpuratus sequences and 16sequences did not return any hits (Table S2). We then performed ananalysis of protein domains and domain architectures derived fromidentified proteins (Figs 4 and 5). As a result, we obtained 68 uniquedomain architectures composed of 68 distinct domain types. EGF-like and SRCR domains occurred most frequently in uniquearchitectures, six and five times, respectively. Significant portionsof domain architectures were composed of only one domain: therewere 29 single-domain, eight two-domain and 30 multi-domainarchitectures. The most complex architectures were observed inHYR/EGF/CCP (HEC) domains containing protein, consisting of53 domains and six distinct domain types; and in coelomotrypsin(CLTR), consisting of 14 domains and six distinct domain types.Combinations of sequence similarity search and domain analysisenabled us to classify proteins into 12 categories according to theirputative molecular function terms (Fig. 3B). The exceptions werenine proteins that could not be classified and were grouped as‘uncharacterized secretory soluble’.

CF proteome includes novel proteinsThere were four uncharacterized secretory soluble proteins withoutany detectable similarity and recognizable domains: CHUPA(CHU), KARTESH (KASH), wound associated starfish peptide(WASSP) and TripleWG protein. CHU, KASH and WASSP are

short polypeptides comprising predicted N-terminal signal peptides.Searches of available transcriptomes of Echinodermata usingTBLASTN identified close homologs in starfish (Asteroidea) andsea urchins (Echinoidea) for CHU (Fig. 6A), whereas KASH andWASSP appear to be specific to Asteroidea (Fig. 6B,C). Both CHUand KASH peptides share some structural similarity: the N-terminalregions contain stretches of acidic residues followed by dibasicconvertase cleavage sites, which indicates that this part may be anacidic spacer peptide, and both peptides bear highly conserved four-cysteine residue motifs, but with different cysteine spacing. All ofthese peptides were identified only in the echinoderms, and theirrelationships with peptides identified in other phyla are unclear.

TripleWG is a 1001-residue protein named on account of twopredicted domains arranged in tandem and repeated three times.These domains are: ∼130 amino acid residues with a conservedWYR motif, and ∼190 amino acid residues with a conserved GWmotif. Several remote homologs were detected for each predicteddomain among invertebrates and lower chordates (Fig. S25);however, the observed domain organization appears to be uniqueto echinoderms.

Injury significantly affects the CF proteomeTo discriminate putative proteins with injury-related production, thelist of all LC-MALDI identified CF proteins was split up over theseparate injury types and time points (Fig. 7A). The PW treatmentyielded 48 identifications before injury (CPW) and 62identifications 6 h post-injury (PW6h). The BL treatment yielded42 identifications before injury (CBL), 62 identifications 6 h post-injury (BL6h) and 37 identifications 72 h post-injury (BL72h).Comparing these sets, eight proteins were common to both PW6hand BL6h, while 20 proteins were exclusively associated withBL6h, and only six with PW6h. Of these 34 proteins with injury-related production, 13 were identified with a single PSM. The mostapparent case of a protein identified uniquely and abundantly inboth types of trauma was the ZP domain containing protein-1(ZPD1). There was only one unique protein specific to the BL72hgroup when compared with both PW and BL trauma, namely thechupa-2 peptide (CHU2). It is noteworthy that WASSP wasidentified in both PW6h and BL72h, indicating delayed

Secretory soluble

Partial atN-terminus

Intracellular GPI-anchored

Single-passtransmembrane

ASignal transduceractivity

Protein binding

Transmembranereceptor activity

Extracellular matrixstructural constituent

Uncharacterized secretorysoluble

Actin binding/cytoskeleton

Peptidase inhibitoractivity

Lipid transporter activity

Carbohydratebinding

Pattern recognitionreceptor activity

Hydrolaseactivity

Oxidoreductaseactivity

Binding

61

9

7

8

4

4

3

8

11

13

8

9 436

9

9

6

B

Fig. 3. Characterization of the coelomic fluid proteome. Pie charts illustrating predicted cellular location (A) and putative functional terms (B) of 91 identifiedproteins. Each protein was assigned to only one functional group.

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upregulation of this peptide following blood loss. Most of theproteins identified in control groups were within an overlap of 27proteins across all groups. Thus, our data indicate that BL injurycaused more prominent qualitative changes in CF composition,revealed by the identification of three times more unique proteinsthan in PW.

We used a G-test to compare proportions of observed andexpected spectral counts for proteins overlapping between controland experimental groups. Differences were significant comparingPW6h with CPW (G=139.1, d.f.=43, P<0.001), BL6h with CBL(G=146.3, d.f.=36, P<0.001) and BL72h with BL6h (G=133.6,d.f.=33, P<0.001). Subsequently, we used the McNemar test for

1 * 75

EGF (EGF-like peptide)

1 * 130

CRH (CRH-type peptide)

? * 301GRAN (Progranulin)

Signal transducer activity

1 * 129

SAMA (Serum amyloid A)

1 * 121

LYSTAR1 (Ly6-like protein-1)

1 * 124

LYSTAR3 (Ly6-like protein-3)

1 * 139

LYSTAR4 (Ly6-like protein-4)

1 * 126

LYSTAR2 (Ly6-like protein-2)

1 * 436N C

RGMA (Repulsive guidance molecule A)

1 * 321

VREP (vWFC-related protein)

1 * 304

MURP (Mucin-related protein)

Extracellular matrix structural constituent

1 * 1418

CALP1 (Collagen alpha-like protein-1)

CALP2 (Collagen alpha-like protein-2)

1 ? 1856

CALP3 (Collagen alpha-like protein-3)

1 ? 824

Cardohydrate binding

1 * 106

SUEL (SUEL-related protein)

1 * 165

CTL1 (C-type lectin-1)

CTL3 (C-type lectin-3)

? * 115

1 * 169

CTL2 (C-type lectin-2)

1 * 177

CTL4 (C-type lectin-4)

1 * 281

TACH (Tachylectin-like protein)

? * 319

FURP (Fucolectin-related protein)

FREP (Fibrinogen-related protein)

1 * 318

1 * 158

LPMO (LPMO-related protein) CTL5 (C-type lectin-5)

? * 110

CTL6 (C-type lectin-6)

? * 131

Pattern recognition receptor activity

1 * 287

ECU1 (EGF and CUB domain containing protein-1)

1 * 402

FASR (FA58C/SRCR-related protein)

1 * 446

ERP (EGF repeat-containing protein)

1 * 694

ECU2 (EGF and CUB domain containing protein-2)

1 * 798

ZPD1 (ZP domain-containing protein-1)

1 * 1529

SSRR (Soluble SRCR-related protein)

1 * 411

FASR2 (FA58C/SRCR-related protein-2)

1 * 1113

SSRR2 (Soluble SRCR-related protein-2)

HEC (HYR/EGF/CCP domains containing protein)

1 * 3544

1 * 687

HEBL (Heme-binding-like protein)

1 * 125

TSR1 (TSR-containing protein-1)

1 * 284

ENDL (Ependymin-like protein)

Protein binding

1 * 119

IGR1 (Immunoglobulin-related protein-1)

TSR2 (TSR-containing protein-2)

? * 276

VTP (VWC/TY domains-containing protein)

? * 648

1 * 214IGR2 (Immunoglobulin-related protein-2)

* 7051

SRCR (SRCR-related protein)

* 7661

LRIG (LRR and IG domain-containing protein)

* 7981

ZPD2 (ZP domain-containing protein-2)

Transmembrane receptor activity

CCRP (Complement control-related protein)

1 * 440

IGR3 (Immunoglobulin-related protein-3)

1 * 601

SUSD (SUSD-related protein)

* 11381

CORP (Collagen-related protein)

1 * 386

1 * 84

CHU1 (Chupa-1)

Uncharacterized secretory soluble

1 * 215DUF2778

DUF1 (DUF2778)

TWG (TripleWG protein)

* 1001RPT 2 RPT 1 RPT 2 RPT 1 RPT 21 RPT 1

DUF2 (DUF2778-2)

* 131DUF2778?

1 * 79

CHU3 (Chupa-3)

1 * 87

CHU2 (Chupa-2)

1 * 90

KASH1 (Kartesh-1)

1 * 79

KASH2 (Kartesh-2)

1 * 66

WASSP (Wound associated starfish peptide)

1 * 210

AREL (Astreelin)

* 2141

AVIL (Avidin-like protein)

Binding

* 4091

ALYS (Asterlysin)

1 * 149

CALM (Calmodulin)

Fig. 4. Themolecular toolkit of coelomic fluid: domain organization of proteins identified in cell-free coelomic fluid of the common starfish, A. rubens.Protein backbone and domain symbols are not shown to scale and indicate the domains’ order and approximate position.

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determination of differentially abundant proteins and illustratedinjury-related changes by means of a heat map depicting both PAIand putative functional terms (Fig. 7B). We identified fourdifferentially abundant proteins in the PW6h group and eightproteins in the BL6h group, with an overlap of two proteins. Of note,by 72 h post-injury, the BL group had downregulated six proteinsand 18 of 20 proteins unique to the BL6h group had disappeared.Comparison of BL72h with CBL (no difference, G=30.7, d.f.=27,P=0.28) revealed that tendency to return to control values wasobserved by 72 h post-injury, with the exception of three uniqueproteins – WASSP, KASH2 and CHU2 – which actually showed

delayed upregulation. In addition to the components showingdifferential regulation, there were abundant proteins unaffectedacross all injury types. The most apparent cases of these are mucin-related protein (MURP), peptidoglycan recognition protein(PGRP), the NlpC/P60 domain containing protein (NLPC) andLy6-like protein-4 (LYSTAR4).

HPLC analysis supports changes caused by injuryBoth PWand BL sets of pooled cell-free CF samples showed nine to10 major protein peaks, clearly identifying the most abundant CFproteins (Fig. 8A,B). The 10 most abundant HPLC peaks were

Hydrolase activity

1 * 174

NLPC (NlpC/P60 domain containing protein)

1 * 357

PTX (Plancitoxin-like protein)

1 * 1547

CLTR (Coelomotrypsin)

1 * 155

LYSO (Lysozyme)

1 * 195

PGRP (Peptidoglycan recognition protein)

CATB (Cathepsin B)

1 * 324

Lipid transporter activity

1 * 1596DUF1943

VTG1 (Vitellogenin-1)

1 * 1930DUF1943

VTG2 (Vitellogenin-2)

1 DUF1943 DUF1081 * 4969

ALPR (Apolipoprotein-like protein)

ALPH (Apolipophorin-like protein)

1 DUF1943 DUF1081 * 3560

1 * 692

MCOX (Multicopper oxidase)

1 * 210

SOD (Superoxide dismutase)

1 * 1115

HEPH (Hephaestin-like protein)

Oxidoreductase activity

Peptidase inhibitor activity

1 * 80

KUTZ (Kunitz-type serine protease inhibitor)

1 * 99

BMSP (BMSP-related protein)

1 * 83

KAZ1 (Kazal-type serine protease inhibitor-1)

1 * 81

KAZ2 (Kazal-type serine protease inhibitor-2)

1 * 168

WAPL (WAP-like protein)

1 * 147

TILT (Putative TIL-type protease inhibitor)

1 * 178

COXI (Carboxypeptidase inhibitor)

1 * 224

TIMP (Metalloproteinase inhibitor)

1 * 1644

ALPI (Asterias large peptidase inhibitor)

TIRP (TIMP-related protein)

1 * 151

1 * 78

KAZ3 (Kazal-type serine protease inhibitor-3)

WAPR1 (WAP-related protein-1)

1 * 85

WAPR2 (WAP-related protein-2)

1 * 73

Actin binding/cytoskeleton

1 * 151

COFL (Cofilin-like protein)

1 * 194

TGLN (Transgelin)

1 * 284

TPMZ (Tropomyosin)

1 * 376

ACTC (Actin, cytoplasmic)

Key to 68 detected domain types

- Serum amyloid A

- C8 (domain containing 8 conserved cysteine residues)

- Tectonin beta propeller repeat

- Thyroglobulin type I repeats

- Calponin homology domain

- Tissue inhibitor of metalloproteinase

- CUB (domain first found in C1r, C1s, uEGF and bone morphogenetic protein)

- Reeler

- Complement control protein

- Fibrillar collagens C-terminal

- Repulsive guidance molecule N-, C-terminal

- SUEL-type lectin

- Corticotropin-releasing factor

- Beta-microseminoprotein

- Deoxyribonuclease II

- PAN_AP (divergent subfamily of APPLE domains)

- Tropomyosin

- Kazal type serine protease inhibitor

- Ly-6 antigen/uPA receptor-like

- Thrombospondin type 1 repeats

- von Willebrand factor type C domain

- Actin depolymerisation factor/cofilin-like

- Leucine-rich repeat

- Multicopper oxidase

- Destabilase

- Superoxide dismutase

- Mucin-2 protein WxxW repeating region

- Collagen triple helix repeat

- Type IV collagen C4 domain

- NlpC/P60 papain-like cysteine peptidase

- Aerolysin toxin

- Lytic polysaccharide mono-oxygenase

- Kringle domain

- AMOP (adhesion-associated domain present in MUC4 and other proteins)

- SOUL heme-binding

- eel-Fucolectin Tachylectin-4 Pentaxrin-1

- Trypsin Inhibitor-like cysteine rich domain

- Agrin N-terminal domain

- Zona pellucida domain

- Lipoprotein N-terminal domain - WSC (yeast cell wall integrity and stress response component proteins)

- Epidermal growth factor-like domain

- Scavenger receptor cysteine-rich

- Actin

- Ependymin

- von Willebrand factor type D domain

- Trypsin-like serine protease

- C-type lectin

- Granulin

- Peptidoglycan recognition protein

- EF-hand, calcium binding motif

- Immunoglobulin

- Avidin

- Fibrinogen C-terminal domain

- BPTI/Kunitz family of serine protease inhibitors

- Coagulation factor 5/8 C-terminal

- Low-density lipoprotein receptor class A

- Four-disulfide core or WAP

- Domains of unknown functionDUF2778DUF1943 DUF1081- Internal repeatRPT

- Signal peptide

- Transmembrane region

- Stop codon *

- GPI anchor signal sequence

Predicted sequence features

- Peptidase family C1 propeptide

- Papain family cysteine protease

- Nematode cuticle collagen N-terminal domain

- Pentraxin / C-reactive protein / pentaxin family

- Nidogen-like domain

- Putative ephrin-receptor like

- HYR (hyalin repeat domain)

Fig. 5. Domain organization of coelomic proteins and key to detected domain types.

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collected and subjected to digestion and MS/MS analysis. Of these,nine protein peaks were identified: MURP, Asterias large peptidaseinhibitor (ALPI), PGRP, Kunitz-type serine protease inhibitor(KUTZ), WAP-like protein (WAPL), CTL1, TIMP-related protein(TIRP), NLPC and vitellogenin-1 (VTG1). As expected, theseproteins were among those with high PAI values determined by thequantitative proteomic approach (Fig. 7B).HPLC profiles of control and injured starfish were substantially

different in peak intensities (Fig. 8A,B). Injury resulted in 1.8- and2.4-fold increases of total peak area for PW and BL, respectively.Injury-related changes in both the PW and BL sets, monitored byabundant components, showed similar trends in accordancewith thequantitative proteomic data. Thus, HPLC analysis showed a goodagreement with data obtained by a quantitative proteomic approach,independently revealing the most abundant CF proteins and theirrelative changes.

Both types of injuries cause increases in coelomocyteconcentrationIn order to correlate proteomic and cellular responses, we examinedthe effect of PW and BL injury on the concentration of circulatingcoelomocytes. Time-dependent changes of the coelomocytes’concentration in response to injury are summarized in Fig. 8C. At6 h after PW, the coelomocytes’ concentration showed a significant5-fold (P=0.002) increase from 1.52±1.12×106 ml−1 to 7.50±4.78×106 ml−1. Notably, at 6 h after BL, the coelomocytes’concentration significantly increased 4.2-fold (P=0.012), from0.44±0.36×106 ml−1 to 1.86±1.28×106 ml−1, followed by a 3.5-fold decrease (P=0.012) to 0.52±0.90×106 ml−1 by 72 h post-injury. These data show that proteomic responses and increases incell concentration are synchronous events, suggesting a functionalrelationship between these processes.

DISCUSSIONHere, we provide an overview of the proteins comprising cell-freeCF of the common starfish, A. rubens. To gain insight into theCF proteome, we paid special attention to isolation of cell-free CF.We utilized needle sampling without aspiration, gentle cellcentrifugation, and filtration of the supernatant to avoidcontamination by intracellular proteins. By this approach we aimedto obtain as accurate as possible a set of extracellular proteins that areactually present in the CF.

The main goal of this study was to analyze early responses ofCF to injury utilizing two traumatic treatments. Changes in CFcomposition revealed in injured starfish at both the cellular andproteomic levels for both types of injuries suggest the importance ofboth cellular and humoral components in early responses to injury.We found that, in spite of the vast loss of whole CF after BL injury,the proteomic response was more pronounced in BL6h than inPW6h, as revealed by both the LC-MALDI and HPLC approaches.We suppose that a vast loss of the entire CF and depletion of thecoelomocyte depot are major factors responsible for the observedproteomic changes after BL injury. Significant post-traumaticincreases in coelomocyte concentrations for both types of injuryindicate that a large number of coelomocytes are withdrawn fromcirculation in normal state, and imply the existence of mechanismseffectively regulating recruitment of cells into the circulation.

Through this work, we showed that PW and BL may share acommon core of injury-responsive proteins. Identification ofproteins common to both types of injuries is important, as theysuggest underlying general mechanisms of response to injury,whereas identification of proteins specific to a particular injury maysuggest activation of different physiological processes contributingto functional discrimination between PW and BL. In the followingsections we will discuss key proteins influenced by injury and

A

B

C

Signal peptide Acidic stretch

Signal peptide Acidic stretch

Signal peptide

Asterias forbesiMarthasterias glacialis-1Marthasterias glacialis-2Leptasterias sp.-1Leptasterias sp.-2Pisaster ochraceus-1Pisaster ochraceus-2Pisaster ochraceus-3Strongylocentrotus purpuratus-1Strongylocentrotus purpuratus-2

Asterias forbesiAsterias amurensisMarthasterias glacialisPisaster ochraceusHenricia sp.Patiria miniataPatiria pectinifera-1 Patiria pectinifera-2 Peribolaster folliculatus

Asterias amurensisMarthasterias glacialisLeptasterias sp.Pisaster ochraceus

Fig. 6. Multiple sequence alignments of three peptides with homologs identified in available transcriptomes of echinoderms. (A) CHUPA, (B)KARTESH and (C) WASSP. Positions of predicted signal peptide, tentative acidic spacer and dibasic convertase cleavage sites are shown in gray, blue and redboxes, respectively. Note conservation of cysteine residues highlighted in yellow. Sequence identities relative to the first A. rubrens sequence are shownon the right. Residues are colored according to physicochemical properties at a 60% identity threshold.

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suggest hypotheses about their potential roles in the physiology ofstarfish.

Pattern recognition receptorsThe largest fraction of the CF proteome is represented by patternrecognition receptors. As echinoderms, starfish lack an acquiredimmunity, relying on the presence of pattern recognition receptorsfor discriminating and eliminating pathogens (Smith et al., 2010).The carbohydrate binding proteins and C-type lectins in particularare key molecules of the echinoderm immune system. We detectedfour C-type lectins and fucolectin-related protein to be upregulatedafter injury. The identified C-type lectins share significantsimilarities with N-acetylgalactosamine-specific lectins from thestarfish Asterina pectinifera (Kakiuchi et al., 2002). Notably, onlyCTL1 showed downregulation in both injury types. It is tempting toassume that CTL1 is consumed during cellular clot formation inresponse to injury. Induction of cell aggregation by C-type lectin intunicates has been demonstrated (Matsumoto et al., 2001). It isnoteworthy that amassin, a CF protein mediating calcium-dependent clot formation and occupying approximately 1% oftotal plasma protein in sea urchins (Hillier and Vacquier, 2003), wasnot detected in our samples. The absence of amassin is remarkable,because clotting in starfish is a calcium-dependent process(Kanungo, 1982), and this may indicate that starfish and seaurchins follow different pathways in clot formation.

Lytic polysaccharide monooxygenase-related protein (LPMO) isunique to BL injury. LPMOs are primarily bacterial and fungalcellulose degrading enzymes (Forsberg et al., 2014). Some of theLPMO-related proteins have lost their enzymatic activity butretained their carbohydrate binding capacity, such as chitin-binding protein from Streptomyces (Schnellmann et al., 1994),suggesting a possible role in carbohydrate recognition for starfishLPMO.

Other important components of the echinoderm immune systemare SRCR-domain containing proteins, which are also wellrepresented in the starfish CF proteome. Sea urchin coelomocytesexpress a highly complex set of SRCR genes with pronouncedvariability in expression levels (Pancer, 2000). In A. pectinifera,membrane and shed soluble forms of the SRCR-related proteinApSRCR1 play the role of opsonins in innate immunity (Furukawaet al., 2012). FASR proteins in addition to SRCR domains alsocontain a coagulation factor 5/8 C-terminal domain that is implicatedin cell adhesion and carbohydrate binding (Baumgartner et al., 1998).

The EGF and GUB domain-containing protein ECU2 wasaffected only by BL injury. This protein resembles bonemorphogenic protein 1 (BMP1) owing to the presence of bothEGF and GUB domains. A role of BMP1 in intestine regeneration inthe sea cucumber Holothuria glaberrima has been demonstrated(Mashanov et al., 2012). The CUB domains are represented inextracellular and developmentally regulated proteins, where theyperform roles in protein recognition (Bork and Beckmann, 1993).EGF repeat-containing protein ERP resembles fibropellins due tothe presence of a long stretch of EGF repeats. Fibropellins are

CPW48

PW6h62

BL6h62

CBL42

48

6 20

228

0

2 3

1

070

27

AC

PW

PW

6hC

BL

BL6

hB

L72h

PW

6hB

L6h

BL7

2hB

0.5 1.5 2.5

PAIDUF1SUSDLRIGCCRPCRH

LYSTAR4TILTTIMPECU1SSRRCTL3SODNLPCIGR2SRCRIGR3

LYSTAR2LYSTAR1LYSTAR3

RGMAEGF

KAZ2KAZ1

WAPR1HECTACHCTL2CTL1HEPHMCOXMURPAREL1

CALM1

CHU2DUF2

KASH2WASSPCORPGRANSAMATSR1KAZ3BMSPSSRR2FASRPLTXLYSOPGRPCATB

CALP3TWG

KASH1CHU3VREPVTPIGR1HEBLALPI

WAPR2FASR2ECU2FURPFREPLPMOVTG2ALPHALPR

CALP1CALP2AVILALYSACTCTPMZTGLNCHU1ZPD2TSR2ENDLKUTZWAPLCOXITIRPERPZPD1CTL4SUELCTL5CTL6VTG1CLTRCOFL Actin binding/cytoskeleton

Hydrolase activity/serine-type endopeptidase activityLipid transporter Pattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptorPattern recognition receptor Peptidase inhibitorPeptidase inhibitorPeptidase inhibitor Peptidase inhibitor Protein binding/cell adhesionProtein binding/complement activationTransmembrane receptor Uncharacterized Actin binding/cytoskeletonActin binding/cytoskeletonActin binding/cytoskeletonBindingBindingExtracellular matrix structural constituent Extracellular matrix structural constituent Lipid transporter Lipid transporter Lipid transporter Pattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor Pattern recognition receptorPeptidase inhibitor Peptidase inhibitor Protein bindingProtein binding Protein binding Protein bindingUncharacterized Uncharacterized Uncharacterized Extracellular matrix structural constituent Hydrolase activity/cysteine-type endopeptidaseHydrolase activity/cytolysisHydrolase activity/cytolysisHydrolase activity/toxin activity/apoptotic processPattern recognition receptorPattern recognition receptorPeptidase inhibitor Peptidase inhibitor Protein binding/complement activationSignal transducer activity/chemoattractant activitySignal transducer activity/growth factor activityTransmembrane receptor Uncharacterized Uncharacterized Uncharacterized Uncharacterized Binding Extracellular matrix structural constituent Oxidoreductase activity/ferroxidase activityOxidoreductase activity/ferroxidase activityPattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor/carbohydrate bindingPattern recognition receptor Peptidase inhibitor Peptidase inhibitor Peptidase inhibitorProtein binding Protein bindingSignal transducer activitySignal transducer activitySignal transducer activitySignal transducer activityTransmembrane receptor/cell adhesionTransmembrane receptor Transmembrane receptor Hydrolase activity/cytolysisOxidoreductase activity/antioxidant activityPattern recognition receptor/carbohydrate bindingPattern recognition receptor Pattern recognition receptor Peptidase inhibitor Peptidase inhibitor Signal transducer activitySignal transducer activity/neuropeptide hormoneTransmembrane receptor/cell adhesionTransmembrane receptor/cell adhesionTransmembrane receptor/cell adhesionUncharacterized

+ ++ +

+

+ ++ ++ +

+ +

+ ++ ++

+++++++++++

++++

+++++++

++

++++

+

+ ++

+

+

Fig. 7. Overview of injury-related changes in A. rubens coelomic fluidproteome. (A) Venn diagram depicting overlap between proteomes of twosimilarly processed sets of starfish subjected to puncture wound (PW6h) andblood loss (BL6h). Control samples are indicated by CPW and CBL. (B) Leftpanel: heat map of protein abundance index (PAI) values showing the relativeabundance of the coelomic fluid proteins. Right panel: differentially abundantproteins according to the McNemar test and proteins defined as upregulatedowing to their exclusive identification after treatment (plus signs). Cells shadedwith red denote upregulation; cells shaded with blue denote downregulation.

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components of the extracellular matrix that play a role in sea urchinembryo development (Bisgrove et al., 1991).An interesting finding was a pair of ZP domain-containing

proteins – ZPD1 and ZPD2 – sharing only 29% identity to eachother. Remarkably, ZPD1 is a soluble protein, whereas ZPD2 is atransmembrane receptor. The ZP domain acts as a module forpolymerization of extracellular proteins into filaments or matrices(Jovine et al., 2005), suggesting a function for identified ZPDproteins in recognition and cell adhesion. Thus, a rich repertoire anddifferential regulation of pattern recognition receptors may indicateactivation of non-self-recognition during early response to injury.

Transmembrane receptorsInterestingly, only single-pass transmembrane proteins without anyintracellular signaling domains were identified in the CF proteome.We propose that these proteins are released into circulation from thecell surface by the proteolytic mechanism known as ectodomainshedding, liberating biologically active soluble forms (Hayashidaet al., 2010). The functions of these proteins are probably related tocell adhesion, pathogen recognition and phagocytosis, whereassignal transduction activity appears to be missing. Only ZPD2 ofeight detected transmembrane proteins was upregulated in responseto injury, indicating increased shedding rates of this receptor, whichin turn may imply regulation of cell adhesion and migration in theearly response to injury.An interesting finding was the identification of novel collagen-

related protein (CORP), a single-pass transmembrane type II proteincontaining two C-terminal type IV collagen domains targeted to theextracellular space. The putative function of CORP may be relatedto cell adhesion, similarly to vertebrate transmembrane collagens(Gordon and Hahn, 2010) and collagen IV inDrosophila (Dai et al.,2017).

Lipid transportersWe identified four proteins related to the large lipid transfer protein(LLTP) superfamily (Smolenaars et al., 2007), which contribute todiscrimination between BL and PW injuries. The roles of LLTPsextend beyond the energy store and nutrient functions. Vitellogeninsand apolipophorins are recognized as lipopolysaccharide (LPS)-responsive acute-phase proteins acting in innate immunity throughpattern recognition receptor activity and direct bactericidal action(Zdybicka-Barabas and Cytrynska, 2013; Zhang et al., 2011). Recentstudies have demonstrated that recombinant DUF1943 and vWFD

domains of LLTPs possess pattern recognition, opsonizing andantibacterial activities in zebrafish (Sun et al., 2013a), a bivalvemollusk (Wu et al., 2015) and a scleractinian coral (Du et al., 2017).Thus, upregulation of LLTPs in starfish may imply its involvement indefense and further indicates activation of innate immunity in responseto injury. However, in Drosophila, apolipophorins are involved insignaling as vehicles for lipid-linked morphogensWnt and Hedgehog(Panáková et al., 2005), whereasWnt genes are implicated in intestineregeneration of sea cucumbers (Mashanov et al., 2012; Sun et al.,2013b) and in early development of the starfish Patiria miniata(McCauley et al., 2013). Therefore, a specific transport role of LLTPsin an early response to injury cannot be excluded.

Signal transducersWe also highlight the role of signal transducers in response to injury.Eight proteins were identified as signal transducers circulating in theCF, of which only serum amyloid A was upregulated, contributingto discrimination between BL and PW injuries. Serum amyloid A isa major vertebrate acute-phase protein with chemokine-likeproperties induced in response to infection, inflammationand injury. Potential functions include induction of migration andchemotactic recruitment of immune cells to the site of injury, andinduction of proinflammatory cytokines and extracellular matrix-degrading enzymes (Uhlar and Whitehead, 1999). Closeassociations between intestinal regeneration and expression ofserum amyloid A have been demonstrated in the sea cucumberH. glaberrima (Santiago et al., 2000). Moreover, LPSadministration to sea cucumbers induces both a cellular responseand upregulation of intestinal expression of serum amyloid A(Santiago-Cardona et al., 2003). Thus, upregulation of serumamyloid A in starfish CF may indicate activation of cell migrationand regenerative processes in early responses to PW injury.

An important finding was identification of progranulin processedinto four granulin-like peptides. Granulins are small 6 kDa peptidesderived from one large precursor. Granulins have multiple biologicalroles: they stimulate proliferation and angiogenesis in wounds,support tumor growth and are involved in early embryogenesis(Bateman and Bennett, 1998; Bateman and Bennett, 2009). Granulin-like growth factor secreted by the human liver fluke inducesangiogenesis and accelerates wound healing of mammalian hosttissues in vivo (Smout et al., 2015). Against this background, it will beof great interest to investigate the physiological role of echinodermgranulin-like peptides in the course of response to injury.

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Fig. 8. Injury-related changes revealed by HPLC and cell counting. (A) Chromatograms of pooled coelomic fluid samples before (gray line) and 6 h afterpuncture wound (red line). (B) Chromatograms of pooled coelomic fluid samples (n=8) before (gray line), 6 h (red line) and 72 h (blue line) after blood loss.In A and B, major peaks identified by MALDI-TOF/TOF are marked; the asterisk shows an unidentified peak. (C) Time-dependent changes of coelomocyteconcentrations in injured starfish. The means (squares), medians (lines), 25th to 75th percentiles (boxes) and range (whiskers) for 12 (puncture wound) and8 (blood loss) samples are shown.

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An interesting finding was the diversity of Ly6-like proteins(LYSTAR), relatively abundant in CF, but unaffected by injury. TheLYSTARs are remotely similar to the Ly6/uPAR superfamily. Thissuperfamily includes secreted signaling proteins and receptors withdiverse functions, including protection of cells from complement-mediated lysis (Fletcher et al., 1994), modulation of neuronalexcitability targeting nicotinic acetylcholine receptors (Lyukmanovaet al., 2016; Tsetlin, 2015), cell adhesion andmigration (Alapati et al.,2014; Hänninen et al., 1997), induction of chemotaxis ofhematopoietic stem cells (Selleri et al., 2006), and antimicrobialactivity (Liu et al., 2017). Because acetylcholine is the majorexcitatory neurotransmitter in echinoderms (Devlin, 2001) andaffects mechanical properties of mutable collagenous tissue(Wilkie, 2002), it is tempting to assume that LYSTARs may act asendogenous modulators of acetylcholinergic transmission in starfish.We also report identification of mature corticotropin releasing

hormone-type peptide (CRH), previously discovered in the A.rubens radial nerve transcriptome (Semmens et al., 2016). In spite ofthe involvement of CRH in the stress response in vertebrates (Koob,1999), it was unaffected by injury, suggesting it may play anotherrole in echinoderms.

Extracellular matrix proteinsThree collagen-like proteins were identified 6 h after PW and BLinjury. Collagen is an important component of starfish tissues,playing a major role in determining the mechanical properties of thebody wall (Blowes et al., 2017). However, extracellular matrix(ECM) components should not normally be present in the CF, andits identification may be indicative of ECM degradation. In seacucumbers, degradation of the fibrous collagen by matrixmetalloproteinases (MMPs) occurs during the early stages ofintestine regeneration (Lamash and Dolmatov, 2013; Miao et al.,2017; Quiñones et al., 2002). Cell migration through the ECM isregulated bymembrane-anchoredMMPs, and some of them possesscollagenase activity (Hotary et al., 2000; Quaranta, 2000). Ofparticular interest is that the appearance of collagens was correlatedwith an increase in coelomocyte concentrations after both injurytypes. In A. rubens, consecutive bleedings have been shown tocause a release of coelomocytes from the coelomic epithelium(Vanden Bossche and Jangoux, 1976) and migration of a specificsub-population of small epitheliocytes into the coelomic cavity(Sharlaimova et al., 2014). Degraded collagens detected in humanserum during cancer progression indicate ECM degradation and cellinvasion (Kehlet et al., 2016). Therefore, we propose that theappearance of collagens in CF in response to injury may indicateECM degradation occurring during cell migration into the coelomiccavity.

HydrolasesAnother remarkable group includes six proteins identified ashydrolases. Of these, only cathepsin B and coelomotrypsin areproteases that potentially could be involved in ECM degradation.Cathepsin B is a lysosomal cysteine protease, and in vertebrates it isinvolved in tumor cell proliferation, invasion and angiogenesis(Aggarwal and Sloane, 2014). Increased activity of MMPs throughdegradation of tissue inhibitors of matrix metalloproteinases(TIMPs) by cathepsin B has been demonstrated (Kostoulas et al.,1999).Coelomotrypsin is a novel multidomain serine protease

consisting of an N-terminal set of recognition and protein bindingdomains and a C-terminal trypsin-like domain. Proteins with asimilar domain architecture have been identified in the P. miniata

skeletal proteome by a proteomic approach (Flores and Livingston,2017). The only characterized proteins that have a somewhat similardomain organization are vertebrate secreted proteases such asneurotrypsin, plasminogen activator and plasminogen. All of theseare secreted as inactive zymogens and subjected to proteolyticactivation, and are involved in various processes: neural plasticity(Stephan et al., 2008), ECM remodeling, cell invasion, adhesionand migration (Irigoyen et al., 1999).

Both lysozyme (Sana et al., 2004) and peptidoglycan recognitionprotein have been shown to have antimicrobial and bacteriolyticactivity because both degrade peptidiglycans, major components ofthe bacterial cell wall (Callewaert and Michiels, 2010; Coteur et al.,2007). Antimicrobial activity can also be proposed for NlpC/P60domain containing protein (NLPC), which shares a remotesimilarity with the NlpC/P60 superfamily of phage-associatedbacteriolytic enzymes and bacterial peptidoglycan hydrolasesinvolved in cell wall biogenesis (Anantharaman and Aravind,2003). Remarkably, the N-terminus of NLPC is highly similar to theonly sequenced N-terminal fragment of interleukin-1-like protein(PIR: A61273) purified from CF of A. forbesi (Beck and Habicht,1991). Strong evidence of IL-1-like activity has been demonstratedin vertebrate assay systems, but no analyses have been performed onechinoderms (Beck and Habicht, 1991).

An interesting finding was the identification of PTX in CF, themajor lethal factor from venomous spines of the crown-of-thornsstarfish, Acanthaster planci (Shiomi et al., 2004). PTX is a toxicDNase II able to enter into the cell and induce apoptosis throughDNA degradation (Ota et al., 2006). The protective role of PTX inmucous secretions of A. rubens (Hennebert et al., 2015) is obvious,whereas the presence of PTX freely circulating in the CF maysuggest its involvement in internal defense against eukaryoticpathogens.

Peptidase inhibitorsIn striking contrast to the scarcity of proteases in CF was the highnumber and abundance of peptidase inhibitors. Our CF proteomecontained 13 proteins predicted to function as peptidase inhibitors,including Kunitz, Kazal, WAP, TIL, TIMP, agrin N-terminal andBMSP domains. The WAP-like protein is a novel member of thewhey acidic proteins (WAP) identified in echinoderms for the firsttime. It is noteworthy that WAP-like protein is a major and injury-responsive protein of starfish CF, whereas WAP is a major milkprotein in some mammals. WAP domain proteins are pleiotropicmolecules and, in addition to peptidase inhibitor activity, they havedemonstrated antimicrobial, antiviral and anti-inflammatoryactivities (Scott et al., 2011). WAP domain proteins are alsoassociated with tumor progression and have been recognized asmarkers for several cancers (Bouchard et al., 2006).

Novel multidomain protein ALPI includes EGF, vWFC,thyroglobulin and Kunitz-type domains, which define its putativepeptidase inhibitor and protein binding activities.

The proteome of CF also contains two metalloproteinaseinhibitors: TIMP and TIRP. The latter contains a single agrin N-terminal domain, structurally related to TIMPs. The function ofTIMPs is related to the regulation of the MMPs activity discussedabove. Thus, although MMPs were not identified in our study, theirpresence is suggested by the identification of TIMPs.

An important protein distinguishing PW from BL injury is β-microseminoprotein (BMSP), also known as prostate secretoryprotein PSP94. The BMSP is present in different human mucoussecretions (Weiber et al., 1990), is involved in inhibition of tumorgrowth (Sutcliffe et al., 2014), possesses antifungal activity

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(Edström et al., 2012) and is overexpressed in early stages ofintestine regeneration in the sea cucumber A. japonicus (Zhanget al., 2017). However, the exact molecular function of thisimportant protein is not yet clearly understood.We propose that a rich repertoire of peptidase inhibitors

circulating in the CF are required for wound healing andregeneration, probably by regulating cell migration and ECMremodeling by orchestrating protease activity. However, aninvolvement in innate immunity through direct antimicrobialaction is also possible.

Binding and protein binding categoryThis category includes several important proteins that furthercontribute to differentiation of BL from PW injury. Ependymin-likeproteins are present in vertebrates, invertebrate deuterostomes, andprotostomes (Suárez-Castillo and García-Arrarás, 2007). Theseproteins have been associated with long-term synaptic plasticity(Shashoua, 1991), are overexpressed in human colorectal tumorcells (Nimmrich et al., 2001), are involved in intestine regenerationin the sea cucumber (Suárez-Castillo et al., 2004) and aredownregulated in vertebrate hematopoietic progenitor cells withthe onset of proliferation and differentiation (Gregorio-King et al.,2002). Calcium-dependent involvement of vertebrate ependymin incell-matrix adhesion has also been proposed (Hoffmann, 1994).The only characterized protein having a domain organization

similar to the identified TSR-containing proteins is vertebrateproperdin, which has seven thrombospondin-type repeats.Properdin in involved in innate immunity through activation ofthe complement alternative pathway, pattern recognition receptoractivity, and recognition and clearance of self-apoptotic andmalignant cells (Kemper et al., 2010). It is important to note thatno components of the complement system previously reported inechinoderms (Smith et al., 2010) were detected in our samples.Therefore, the complement system appears to be unaffected orexpressed at undetectable levels under the conditions that we tested.The importance of vWFC-related protein (VREP) in early

responses to BL arises from its domain architecture similaritywith vertebrate brorin, namely two von Willebrand factor Cdomains. Brorin, a member of the chordin family, is involved inneurogenesis in vertebrates as a BMP antagonist (Miyake et al.,2017), whereas chordin shapes the BMP morphogen gradientduring sea urchin embryo development (Lapraz et al., 2009).High affinity of vertebrate avidin to biotin is generally thought to

inhibit bacterial growth, whereas the avidin-like domain of seaurchin fibropellin does not bind to biotin and was adopted foroligomerization (Itai et al., 2005). However, tissue-specificexpression of avidin-like genes during early and late stages ofintestine regeneration in the sea cucumber A. japonicus has beenreported (Ba et al., 2015), suggesting an important role for starfishavidin-like protein in response to BL injury.Asterlysin is a novel starfish protein with a bacterial aerolysin

toxin domain. Aerolysin is a cytolytic pore-forming toxin from agram-negative bacterium Aeromonas hydrophyla that targetseukaryotic cells (Howard et al., 1987), whereas eukaryoticaerolysins serve as defense molecules in Biomphalaria snails(Galinier et al., 2013) or assist in osmotically driven preydisintegration in hydra (Sher et al., 2008).

Actin-binding proteinsThe group of actin-binding proteins was also upregulated after injuryand further contributes to differentiation of BL from PW injury.Intracellular proteins are often considered to be contaminants or tissue

leakage proteins in studies of extracellular proteomes. Because ourCF isolation procedure was intended to produce non-traumaticsampling and gentle cell removal, an alternative interpretation is thatthese proteins may be genuine components of the CF, playing a roledistinct from their intracellular function. Indeed, release of actin uponcell death and formation of actin filaments in body fluids aredetrimental, and an actin scavenger system exists in vertebrates tosequester actin and facilitate its clearance from circulation (Lee andGalbraith, 1992). Thus, cofilin-like protein with actin filamentdepolymerisation activity may be a principal component of actinscavenger systems in starfish, like gelsolin in vertebrates, stabilizingextracellular actin in a globular form.

In contrast, there is strong evidence of direct involvement of actinin invertebrate innate immunity. In Drosophila, extracellular actintriggers a response associated with wounding and dead cellclearance (Srinivasan et al., 2016). Actin-derived antimicrobialpeptides have been identified in A. rubens (Maltseva et al., 2007),and extracellular actin has been shown to mediate antimicrobialdefense in the mosquito Anopheles gambiae (Sandiford et al.,2015). Interestingly, bactericidal activity has been shown to be moreprofound for globular actin, and our identification of the cofilin-likeprotein is in line with these data.

‘Orphans’ among the CF proteomeHigh-throughput transcriptomic and proteomic approaches targetedon non-model invertebrates have often revealed novel proteins thatlack database matches or conserved domains that would allow forspeculation about their functions. Indeed, approximately two-thirdsof differentially expressed genes identified from transcriptomicanalyses in echinoderms are ‘unknown genes’ not included in GOanalysis (Fuess et al., 2015; Sun et al., 2013b). In starfish CF, weidentified eight uncharacterized ‘orphan’ proteins, of which themost intriguing are injury-responsive Chupa, Kartesh and WASSPpeptides. It is noteworthy that novel Chupa and Kartesh peptides arepresent in several isoforms, suggesting some functional divergence.Physiological roles of these peptides currently cannot behypothesized; however, probable functions of small extracellularcysteine-rich proteins are predominantly related to signaling(hormones, growth factors) and binding (enzyme inhibitors,toxins, defensins). Identification of novel proteins withtaxonomically restricted distribution is especially exciting becausethese might be responsible for adaptations specific for starfish, andcan provide a unique opportunity to investigate the physiologicalroles of these ‘orphans’ in early responses to injury.

ConclusionsThe data that we present here provide an overview of the coelomicproteins of the starfish A. rubens, expanding our knowledge ofechinoderm CF proteomes. However, our work clearly has somelimitations. A major source of unreliability is the use of single pooledbiological replicates in proteomic analyses. Given that our quantitativeproteomic findings are based on a single replicates, the results fromsuch analyses should be treated with caution. Nevertheless, our datayielded valuable insights into the process of the response to injury,encouraging further investigations using larger sample sizes andrepeated measures over a longer period of time, targeting moreconfident and precise delineation of the injury-related changes.

Through this work, we revealed a list of injury-responsive proteinspotentially involved in defense, cell migration and wound healing,and demonstrated dramatic effects of injury on the CF proteome.Injury-responsive proteins, such as KUTZ, WAPL, TIRP and VTG1,both abundant and easily detected by HPLC, seem to be suitable

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candidate biomarkers of injury in starfish. Injury-responsive proteinswith no clear homology, such as ALPI, VREP, ECU2 and other‘orphans’, are also of outstanding interest for functional studies.Although our findings suggest that BL is distinguished from PW

by a set of proteins, the predicted functional terms are too general,hindering recognition of injury-specific physiological processes. Thisis a second limitation of our work and a general challenge of studieson non-model invertebrates. Therefore, further experiments onrecombinant protein expression, tertiary structure determination andfunctional activity testing are needed to clarify the physiological rolesof these proteins. Nevertheless, an overlap of both injury-responsiveproteins and general functional terms between injuries could suggestfive common processes playing a role in early responses to injury: (1)activation of innate immunity involving defense and non-self-recognition; (2) regulation of proteolysis by a rich repertoire ofpeptidase inhibitors; (3) activation of proteolysis and degradation ofcollagenous ECM; (4) regulation of cell adhesion and migration; and(5) activation of regenerative processes. Our assumptions about theactivation of regenerative processes is supported by an overlap ofinjury-responsive proteins with those involved in regeneration in seacucumbers, namely β-microseminoprotein, serum amyloid A, andependymin-like and avidin-like proteins. Observed associations ofcancer-related terms with such proteins as WAPL, LYSTARs,GRAN, BMSP, CATB, CLTR and ENDL is not surprising,because carcinogenesis involves both cell migration andtransdifferentiation, which are thought to be important mechanismsof regeneration in echinoderms (Kalacheva et al., 2017). Therefore,our dataset may represent an important tool for discovery of novelproteins involved in regeneration in echinoderms, suggestingimportant targets for future studies. Moreover, observed signs ofinnate immune response call for further studies comparing responsesto immune challenge and wounding, and targeting more precisediscrimination of regeneration-related components.In summary, despite the limitations of this study, our CF

proteome represents an important starting point to understand theunderlying mechanisms of early response to injury in echinoderms,highlights the importance of both ‘orphan’ proteins and proteinsconserved throughout the deuterostomian lineage, and provides theopportunity to investigate the physiological roles of these proteins.

AcknowledgementsWe are grateful to the staff of theWhite Sea Biological Station ‘Kartesh’ of ZoologicalInstitute of the Russian Academy of Sciences for the provided facilities andassistance. We thank Drs Maurice Elphick and Patrick Flammang for sharingassembled transcriptome datasets.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.S.; Methodology: S.S.; Investigation: S.S., D.B., N.S., O.P.;Writing - original draft: S.S.; Writing - review & editing: S.S., O.P.; Visualization: S.S.;Supervision: O.P.; Funding acquisition: O.P.

FundingThis work was supported by the Russian Foundation for Basic Research (project no.15-04-07798).

Data availabilityThe mass spectrometry proteomics data and search database have been depositedat the ProteomeXchange Consortium via the PRIDE partner repository: PXD010228

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.198556.supplemental

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