Metallothionein Gene Family in the Sea Urchin Paracentrotus lividus

26 Pages·2017·3.11 MB·English

International Journal o f
Molecular Sciences
Article
Metallothionein Gene Family in the
Sea Urchin Paracentrotus lividus: Gene Structure,
Differential Expression and Phylogenetic Analysis
MariaAntoniettaRagusa1,*,AldoNicosia2,SalvatoreCosta1,AngelaCuttitta2and
FabrizioGianguzza1
1 DepartmentofBiological,Chemical,andPharmaceuticalSciencesandTechnologies,UniversityofPalermo,
90128Palermo,Italy;[email protected](S.C.);[email protected](F.G.)
2 LaboratoryofMolecularEcologyandBiotechnology,NationalResearchCouncil-InstituteforMarineand
CoastalEnvironment(IAMC-CNR)DetachedUnitofCapoGranitola,TorrettaGranitola,91021Trapani,
Italy;[email protected](A.N.);[email protected](A.C.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:MasatoshiMaki
Received:6March2017;Accepted:5April2017;Published:12April2017
Abstract: Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such
as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and
evolution,weclonedsevenParacentrotuslividusMTgenes,comparingthemtoEchinodermataand
Chordatagenes. Moreover,weperformedaphylogeneticanalysisof32MTsfromdifferentclassesof
echinodermsand13MTsfromthemostancientchordates,highlightingtherelationshipsbetween
them. SinceMTshavemultiplerolesinthecells,weperformedRT-qPCRandinsituhybridization
experiments to understand better MT functions in sea urchin embryos. Results showed that the
expressionofMTsisregulatedthroughoutdevelopmentinacelltype-specificmannerandinresponse
tovariousmetals. TheMT7transcriptisexpressedinalltissues,especiallyinthestomachandin
theintestineofthelarva,butitislessmetal-responsive. Incontrast,MT8isectodermicandrises
onlyatrelativelyhighmetaldoses. MT5andMT6expressionishighlystimulatedbymetalsinthe
mesenchymecells. OurresultssuggestthattheP.lividusMTfamilyoriginatedafterthespeciation
eventsbygeneduplications,evolvingdevelopmentalandenvironmentalsub-functionalization.
Keywords:metallothionein;multigenefamilies;evolution;metal;echinoderms;embryonicdevelopment;
geneexpression
1. Introduction
Metallothioneins (MTs) represent a superfamily of widespread proteins existing of many
organisms, ranging from prokaryotes to vertebrates. The superfamily consists of constitutive and
stress-induciblemembers,withvariablemasses. Itisrichincysteine(Cys)residues(nearly30%oftheir
aminoacidcomposition),andtheresiduesconstitutethemetal–thiolateclusters[1]. MTspossessgreat
affinityforbothessential(zinc,copper,selenium)andxenobiotic(cadmium,lead,mercury)metals,
bindingthemthroughspecificCys-CysandCys-Xxx-Cysmotifs. Usually,Cys-Cysmotifsarelocated
intheC-terminalmoiety(alsoknownas↵-domain), andCys-Xxx-CysmotifsmaptheN-terminal
half(or�-domain). MTsareknowntoexhibitaplethoraofbiologicalfunctions,includingprotection
againstmetaltoxicity,controlofoxidativestressandregulationofphysiologicalhomeostasis[2–5].
Inadditiontotheircentralroleasmetalscavengers,MTsarealsoinvolvedinanumberofcellular
activities,includingcellproliferation[6],differentiation[7,8],apoptosisandimmuneresponse[9,10].
Finally,MTshavealsogainedattentioninbiomedicalstudies,duetotheirproposedinvolvementin
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cancerandneurologicaldiseases[11–15]. MTshavebeenclassifiedintodifferentfamiliesonthebasis
ofstructuralfeaturesandarrangementofCysmotifs[16,17]. Nevertheless,thelargenumberofMTs
discoveredsofarclearlydemonstratestheexistenceofintermediateisoforms,makingtheclassification
ofMTsverychallenging.
Indeed, mechanisms of duplication, convergence and functional differentiation have created
acomplexevolutionaryhistory,whichisdifficulttodefine[18–20].
Throughtheyears,severalstudiesexploringMTsindifferentorganisms(fromvertebrates,suchas
humans,rodents,aves,andamphibians,toinvertebrates,suchasmolluscs,nematodesandinsects)
havebeenreported[17].
Deuterostomesuperphylumdiversifiedaround510millionyearsago(Myr),intoechinoderms,
hemichordates,tunicatesandvertebrates. TheEchinodermataphylumincludesseastars(Asteroidea),
seaurchinsandsanddollars(Echinoidea),brittlestars(Ophiuroidea),seacucumbers(Holothuroidea)
and sea lilies (Crinoidea). Thus, Echinoderms represent a very fascinating phylum since they are
closelyrelatedtoChordates.
AmongEchinodermata,seaurchinMThomologueshavebeenidentifiedinStrongylocentrotus
purpuratus [21], Lytechinus pictus [22], Sterechinus neumayeri and Sphaerechinus granularis [23],
revealing an unusual distribution of Cys motifs. Additionally, the three-dimensional structure
analysisofS.purpuratusMTArevealedthatthisunusualCysmotifdistributioncausedaninverted
architectureofthe↵-and�-domainswithrespecttovertebratestructure[24]. Previously,wereported
the identification of five different MT homologues (PlMT4–8) from the Mediterranean sea urchin
speciesParacentrotuslividus. Twofamilymembers,PlMT7andPlMT8,areconstitutivelyexpressed
andupregulatedinresponsetocadmiumtreatment,whereasPlMT4,PlMT5andPlMT6appeartobe
specificallyswitched-onaftercadmiumexposure[25].
Herein,withtheaimofbetterunderstandingtheevolutionaryrelationships,functionalvariety,
andtheutilizationofMTsduringdevelopment,thegeneorganisationofP.lividusMTswasanalysed
andtheirmRNAexpressionpatternswereunveiled.Particularly,wedeterminedtheexpressionprofiles
andthespatialpatternsofP.lividusMTtranscriptsduringdevelopmentandaftermetaltreatments.
Moreover,exploitingtheadvancesinhomologuesdetectionandhomologyproteinmodelling,
theoretical structure calculation methods were applied. Evolutionary perspectives on MTs in
deuterostomeswereaccomplishedcombiningphylogenyandgenefeatures.
2. Results
2.1. TheMetallothioneinGenesofP.lividus
Theavailabilityoflarge-scaletranscriptionaldatasetsfortheMediterraneanseaurchinP.lividus
allowed us to carry out a transcriptome survey for a comprehensive identification of the MT
homologues. WeperformedBLASTNandTBLASTNsearchesusingMTcDNAsequencespreviously
clonedasqueries[25]andaclusteringanalysisofMTexpressedsequencetags(EST)retrieved.NoMT4,
MT5orMT6sequenceswerefoundinthedatabases,confirmingtheirlowexpression. Collectively,
two MT7 transcript populations differing in length and three MT8 different populations were
retrieved. Theiridentificationwascheckedmanuallyandthematchingsequenceswerereconfirmed
bycomparativeanalysis. TheseresultssuggestthatMT7transcriptsmayderivefromasinglegeneby
alternativesplicingormultiplepolyadenylationsignalsorevenfromtwodifferentgenes. Moreover,
itispossibletohypothesisethepresenceofatleastthreeMT8genes.
In order to identify and isolate the expressed MT genes, total genomic DNA from P. lividus
spermwasextractedandamplifiedusingprimerpairsselectedasdescribedinMaterialsandMethods.
Theamplifiedproductswerecloned,sequencedandanalysed. Fourgenomicclones,codingforMT4,
MT5,MT6,MT7andthreediverseclonescorrespondingtoMT8(namedMT8a,b,c)wereobtained.
ThecomparisonbetweencDNAandgenomicsequencesrevealedthatthetranscriptionunitsare
composedbyfourexonsinterruptedbythreeintronsandaredifferentinlength. Thefirsttwointrons
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interruptthecodingsequenceafterthefirstnucleotideofthecodon(phase-1),thelastintronislocated
inthinet3er0rUupTtR t.hTe hcoedgineng esesqturuecntcuer aefsteorf ththe efiPrs.tl nivuidcluesotMidTe ogfe tnhees coadreonre (pprheasseen-1t)e,d thien laFsitg iunrtero1n. iAs llolcoaftetdh em
possiens tshcea 3n′ oUnTicRa. lTshpel igceinnge sstirtuecst,uidreesn otiffi thede Pa.t l5iv-ideunsd MbTy gGeTneasn adrea rte3pr-eesnedntbeyd AinG Ficgounrsee 1n. sAulsl osef qthueemnc es.
0 0
Morpeoosvseers,sa cacnoomnpicaarla stpivliecianng asliytessi,s idbeentwtifeieedn aPt. 5li′v-eidnuds bayn GdTS a.npdu raptu 3r′a-etnuds MbyT AgGe nceosns(eSntrsouns gseyqlouceenncterso. tid
Moreover, a comparative analysis between P. lividus and S. purpuratus MT genes (Strongylocentrotid
diverged 35–50 Myr from the Parechinidae [26]) showed that all possess the same structure [27].
diverged 35–50 Myr from the Parechinidae [26]) showed that all possess the same structure [27].
Nevertheless,intronlengthsandsequencesaredifferentbetweenallhomologousgenes.
Nevertheless, intron lengths and sequences are different between all homologous genes.

Figure 1. Schematic gene structures of the Paracentrotus lividus and Strongylocentrotus purpuratus
Figure 1. Schematic gene structures of the Paracentrotus lividus and Strongylocentrotus purpuratus
metallothioneins (MTs; drawn to scale). The bent arrows indicate the putative transcription start sites
metallothioneins(MTs;drawntoscale).Thebentarrowsindicatetheputativetranscriptionstartsites
(TSS). Numbers under schemes indicate base pair numbers of exons and introns; boxes represent
(TSS).Numbersunderschemesindicatebasepairnumbersofexonsandintrons; boxesrepresent
exons: white boxes indicate untranslated regions, and coding regions are coloured. The grey diamond
exons:whiteboxesindicateuntranslatedregions,andcodingregionsarecoloured.Thegreydiamond
indicates one N stretch in the SpMTB1 intron.
indicatesoneNstretchintheSpMTB1intron.
In silico predictions showed two polyadenylation sites in the MT7 gene (score 0.876 and 0.898)
wInhiscihli ccoouplrde deixcptiloanins sthhoew perdestewncoep oofl ytawdoe nMyTla7t iomnRsNiteAs isnpethcieesM dTi7ffegreennet (isnc olreeng0.t8h7 6duanridng0 .e8m98b)rwyoh ich
coulddeevxeplolpaimnetnhte. presenceoftwoMT7mRNAspeciesdifferentinlengthduringembryodevelopment.
MTM8bTa8nb danMdT M8cT8sch oshwoewdeda papprporxoixmimaatetelyly tthhee ssaammee lleennggtthh aanndd 9944%% ididenetnittyit. yB.oBtho tghegneesn ecosnctoanint ain
136a13d6d aitdiodnitaiolnbapl sbpins tinh ethfier fsitrsitn itnrtornona nandda a4 47766--bbpp ddeelleettiioonn iinn tthhee sesecocnodn dinitnrotrno wniwthi trhesrpeescpt etcot MtoTM8aT. 8a.
In alignable sequences, the identity between MT8a and MT8b is 97%, higher than the identity with
Inalignablesequences,theidentitybetweenMT8aandMT8bis97%,higherthantheidentitywith
MT8c (93%). The MT8a and MT8b-expressed sequences show 98% of identity between them and 95%
MT8c(93%). TheMT8aandMT8b-expressedsequencesshow98%ofidentitybetweenthemand95%
with MT8c, differing for the presence of a simple AT-rich region in the 3′ UTR.
withMT8c,differingforthepresenceofasimpleAT-richregioninthe30 UTR.
2.2. Predicted 3D Structural Model of P. lividus MTs
2.2. Predicted3DStructuralModelofP.lividusMTs
On the basis of the computational analysis, we determined the key features of MT homologues
Onthebasisofthecomputationalanalysis,wedeterminedthekeyfeaturesofMThomologues
in P. lividus. As previously highlighted [25], only MT7 and MT8 follow the MT family 4 rule
in P. lividus. As previously highlighted [25], only MT7 and MT8 follow the MT family 4 rule
(Echinoidea: IPR001396; [17]): P-D-x-K-C-[V,F]-C-C-x(5)-C-x-C-x(4)-C-C-x(4)-C-C-x(4,6)-C-C located
(Echinoidea:IPR001396;[17]): P-D-x-K-C-[V,F]-C-C-x(5)-C-x-C-x(4)-C-C-x(4)-C-C-x(4,6)-C-Clocated
near the N terminus. Other isoforms instead have divergent amino acid sequences and in particular
neartheNterminus. Otherisoformsinsteadhavedivergentaminoacidsequencesandinparticular
MT4 and MT6 have a cysteine pattern that is slightly different.
MT4andMT6haveacysteinepatternthatisslightlydifferent.
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The MTs are organized in a N-domain and a C-domain bearing a pattern of conserved Cys
The MTs are organized in a N-domain and a C-domain bearing a pattern of conserved Cys
residues required for binding bivalent metal ions [17]. As occurred in S. purpuratus MTs, Cys-Cys
residues required for binding bivalent metal ions [17]. As occurred in S. purpuratus MTs, Cys-Cys
motifs,typicaloftheC-domaininmammals,arelocatedintheN-domain.Multiplesequencealignment
motifInst,. Jt. yMpoli. cSacil. 2o01f7 , t1h8,e 8 1C2 - domain in mammals, are located in the N-domain. Multiple 4s oef q25u ence
(MSA)analysisofP.lividusandS.purpuratusMTs(Figure2)showedthat,inadditiontoconserved
alignment (MSA) analysis of P. lividus and S. purpuratus MTs (Figure 2) showed that, in addition to
CyspatteTrnh,e tMheTas cacreep otergdaanmizeindo ina cai dNs-sduobmsatiintu atinodn sa dCo-dnoomtaailnw baeyasripngo sas epsasttseirmn iolaf rcponhsyesrivceadl cChyesm ical
conserved Cys pattern, the accepted amino acids substitutions do not always possess similar physical
featurreessi.dWuees arregquueirtehda ftosru bcihndchinagn bgievsamlenigt hmtentoalt isounpsp [o17rt].t hAes soacmcuerrsetdru icnt uSr. epuanrpdurcaotuusl dMaTltse, rCbyiso-cChyesm ical
pchroepmemirctoaiteli fsfs,e, aatltyluoprwiecsain.l WgofMe taThreig nuCve-od tlohvmaetma isneu ncitnh i ncmhdaaminffgmeerasel snm,t aipgraeht thl onwcoaatty essdu. pinp otrhte thNe- dsoammaei ns.t rMucutlutirpel ea nsdeq cuoeunlcde alter
biochaelimgnicmael nptr (oMpSeArt)i easn,a alyllsoisw oifn Pg. MlivTid uins vaonldv eSm. peunrpt uirna tduisf fMerTesn (tF pigautrhew 2a) yshs.o wed that, in addition to
conserved Cys pattern, the accepted amino acids substitutions do not always possess similar physical
chemical features. We argue that such changes might not support the same structure and could alter
biochemical properties, allowing MT involvement in different pathways.

FFiigguurree 22.. MuMltuipltliep sleeqsueeqnuceen acleignamligennmt (eMntSA(M) oSfA P). loivfidPu.s laivniddu Ss. paunrdpuSra.tupsu rMpuTr saetuqsueMncTess. eIdquenetnictieess.
Iadnedn tciotinesseravnadtivcoe nssuebrsvtaittuivtieonsus basrtei tiunt iroends foanret. iRnerde dshfaodnint.g Rreepdressheandtisn igderneptirtyes aemntosnigd eanllt isteyquamenoc nesg.
aDllostse Fqdiugeeunnroect ee2s .g .MaDpuolstt.is pNdleoe snne-oqctuoeenngscaeepr vasl.aigtNinvomen es-nuctob (nsMstieStruAvt)ai ootfin vPse . alsirvueib diusnts i btaulnatdcio kSn .f spounarrtpe.u irnatubsl aMckT fsoeqnut.ences. Identities
and conservative substitutions are in red font. Red shading represents identity among all sequences.
IInn oorrDddoeetrsr dttooen ooobtbett agaiainpns s.s oNommonee-c ioinnnddseiirccvaaatttiiiovoenn s ouonbns tssittturrutuioccnttusu rareree c cinhh abanlnagcgkee sfso,,n wwt. ee ccoommppuutteedd tthhee sseeccoonnddaarryy eelleemmeennttss
and derived the 3D structures of P. lividus MTs (PlMTs). Different templates were selected to model
andderivedthe3DstructuresofP.lividusMTs(PlMTs). Differenttemplateswereselectedtomodelthe
the MediItne rorradneera tno osbetaa iunr scohmine iMndTicsa, toionn tohne sbtrauscitsu roef chhaenugreisst, iwcse tcoo mmpauxtiemd itshee csoecnofniddeanryc ee,l epmeernctesn tage
MediterraneanseaurchinMTs,onthebasisofheuristicstomaximiseconfidence,percentageidentity
and derived the 3D structures of P. lividus MTs (PlMTs). Different templates were selected to model
identity and alignment coverage. If required, insertions were modelled ab initio. The generated
and alignment coverage. If required, insertions were modelled ab initio. The generated models
the Mediterranean sea urchin MTs, on the basis of heuristics to maximise confidence, percentage
models were validated by assessing Ramachandran plot analysis and the percentage of residues in
wereidveanlitditya taendd bayligansmseesnsti ncgovReraamgea. cIhf arneqduriarnedp, liontseartnioanlys swiseraen dmotdheellepde racbe nintaitgioe. oTfher egseidneureastedin the
the favoured/allowed region ranged from 91% to 96%. In a manner similar to those described in
favoumreodde/las lwloewree vdalridegatieodn brya ansgseesdsinfrgo Rmam91ac%hatnodr9a6n% p.lotI nanaalymsias nannder thsei mpeilracernttoagteh oofs reesdideuscesr iibne d in
previous studies, PlMTs mainly consisted of coils and turns; while different numbers of helical
previtohue sfasvtouudrieeds/,alPlolMweTds rmegaioinnl yrancogends ifsrtoemd o91f%c otiol s96a%n.d Intu ar nmsa;nwnehri lseimdiliaffre troe nthtonseu mdebsecrrisbeodf ihne lical
ssttrruucctptuurrerevesiso wwusee rrseetu ccdooimmesp,p uPutlteMeddT (s(t thhmrreaeeien ↵lαy-- hhceoelnliicsceiesstse iidnn MoMf TTc4o4,i,l MsM aTTn66d aa ntnuddrn MMs;T Tw88,h, titwlwe ood aiafnfneddre ffnootuu nrru ↵αm--hhbeeelrliiscc eeossf iihnne MMlicTaTl55 aanndd
MMTT77,,s rtrreeusscppteuecrctetisivv weelelyyr)e).. c omputed (three α-helices in MT4, MT6 and MT8, two and four α-helices in MT5 and
MGeTn7e, rreaslplye,c ttihvee lgyl)o. bal structure of each protein resembled that of MTs from other organisms: two
Generally, the global structure of each protein resembled that of MTs from other organisms:
domains Gceonnenrealcltye, dth eb ygl oab afll esxtriubclteu rhei nofg eea. chH porwoteevine rr,e sMemTb4l eadn tdha tM oTf M5 Tpsr fortoemin ost haepr poergaarnedis mtos: tbweo more
twodomainsconnectedbyaflexiblehinge. However,MT4andMT5proteinsappearedtobemore
compdaocmt athinasn c MonTn6ec, t7e da nbdy 8a (flFeigxuibrlee 3h)i.n Tgeh.e H reolwateivveer ,s MolvT4e natn adc cMesTs5i bpilriottye i(nRsS aAp)p oeaf rtehde tCo ybse r emsoidreu es in
compactthanMT6,7and8(Figure3). Therelativesolventaccessibility(RSA)oftheCysresiduesinthe
compact than MT6, 7 and 8 (Figure 3). The relative solvent accessibility (RSA) of the Cys residues in
the folded protein was also calculated. Based on RSA values, sea urchin MTs exhibited a similar
foldedproteinwasalsocalculated. BasedonRSAvalues,seaurchinMTsexhibitedasimilarpattern
the folded protein was also calculated. Based on RSA values, sea urchin MTs exhibited a similar
pattern of solvent accessibility in the Cys, as such residues were found to mainly adopt a buried (RSA
ofsolpvaetntetrna cocfe ssoslivbeinlitt ayccinestshibeilCityy sin, athses Cuychs, ares ssiudcuhe rseswideurees fwouerned fotuonmd taoi nmlyainaldyo apdtoaptb au bruierdied(R (RSSAA< 0.1)
< 0.1) or intermediate (0.1 < RSA < 0.4) conformation. Conversely, exposed and highly exposed
orint<e r0m.1)e doria itnete(0rm.1e<diaRtSe A(0<.1 0<. 4R)ScAo n<f o0r.4m) actoinofno.rmCaotniovne. rCseolnyv,eerxspeloys, eedxpaonsdedh aignhdl yhiegxhplyo seexdporseesdi dues
residues (0.4 < RSA < 1 and RSA > 1 respectively) were found to be strongly underrepresented (Figure
(0.4<reRsSidAue<s (10a.4n <d RRSASA < 1> a1ndre RspSAec >t i1v reelsyp)ewcteivreelyfo) uwnedre tfooubneds ttoro bneg sltyrounngldye urnredperrerespernetseednt(eFdi g(Fuirgeur3e) .
3). 3).

Figure 3. Ribbon diagrams and surface representations of PlMTs. (A) General overview of the 5 PlMTs
Figure3.RibbondiagramsandsurfacerepresentationsofPlMTs.(A)Generaloverviewofthe5PlMTs
generated by homology modelling with cysteine residues labelled in orange. As far as possible, N-
gFeignuerrae t3e.d Ribbybohno mdioalgorgaymsm aondde slluinrfgacwe irtehprceyssetneitnateiorness iodfu PelsMlTabs.e (lAle)d Giennoerraaln ogvee.rvAieswfa orf athsep 5o PsslMibTles,
terminal is on the left and C-terminal is on the right; (B) Relative solvent accessibility (RSA) calculated
Nge-nteerramas tipenedar lcbeisyno tahngoetmh oeof llceoofgrtryae nsmpdooCndd-etielnlrgimn cgiyn swateliiitnsheo crneystsihtdeeuirneiseg ihnrte ;es(aiBdch)u Rpesreo llataetbiinve.el lseodl vinen otraacncegses. ibAisli tfyar(R aSsA p)ocsaslicbullea, tNed-
atesrpmeirncaeln itsa goen othfec olerfrte aspnodn Cd-itnegrmcyinstael iinse orne sthideu reigshint; e(Bac) hReplraottievien s.olvent accessibility (RSA) calculated
as percentage of corresponding cysteine residues in each protein.
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AccordingtothestructuralfeaturesofS.purpuratusMTs(SpMTs)[24]andinsilicoprotein–ligand
bindiAngccosritdeinrge ctoo gtnhiet iostnr,ucptuorsasli bfleeatmureetsa lo–ft hSi.o plautrepucrlautustse rMTstsr u(SctpuMreTss)o [f24P]l ManTds inw esirleicop rperdoitcetiend–.
Tlihgeanadb sbeinncdeinogf ssiptee crieficcogCnyistiorens, ipdousessibinle tmheetCal-–dtohmioalainte ocfluMstTer4 ,st5ruacntdur6esa osfw PelMll Tass twheerep rperseednicceteodf.
aTnhea dadbisteionncea loCf yspserecisfiidc uCeyisn rtehseidNu-edso imn athine oCf-MdoTm6ariani soef tMheTp4o, s5s aibnidli t6y aths awtethlle asse thhoem porleosgeuncees wofe aren
laikdedliytipornoanl eCtyosa crceespidtuvea riina ttiohne sNin-dmoemtaalinb inodf iMngTw6 irtahiosue ttmheo dpiofiscsaibtiiolintsy otfhtahte thtoetsael nhuommboelorgoufebso nwdeerde
dliikveallye nptrocantei otnos .acEcelspetw vhaerriea,tiothnes Nin- manedtaCl -bdinodminaign swoitfhtohuets emhoodmifioclaotgiouness omf atyhel iktoetlayl ennucmombepra sosf
dbiofnfedreednt mdievtaalle–ntht iocalattioencslu. sEtelsrse.where, the N- and C-domains of these homologues may likely
encoAmspasshso dwifnfeirnenFti gmuerteal4–,thfiooulartea ncdlusthterrese. metal–thiolate clusters were identified in the N- and
C-domAsa isnhroewspne citniv FeilgyuorfeM 4T, 7foaunrd aMndT 8t.hTrehee mcluetsatel–rtchoinonlaetcet icvliutisetserbse twweereen itdheenctyifsiteedin ien tthhioe laNte- garnodu pCs-
adnodmtahien mreestpaelcitoivnesliyn otfh MeNT7- aanndd MC-Td8o.m Thaien csluosftMerT c7onannedctMivTit8iews ebreetwcoemenp tahree dcywstietihnteh tehiwolealtl-ed gerfionuepds
SapnMd tThAe. mThetuasl, iaolngso irnit hthme sNto- asnudp eCr-imdopmosaeintsh oefp MolTy7p eapntdid MeTb8a cwkberoen ceosmfrpoamretdh ewseithh othmeo wloeglul-edsewfineerde
aSpppMliTedAa. nTdhuans,a alolggooruisthsmpast itaol asurrpaenrgimempeonstes tohfeC pyoslryepseidputiedsea bmaocnkgbostnreusc tfurorems athnedsree lhaotemdomloegtuale–ss uwlfeurer
calpupstleierds wanedre arnetarlioegvoeuds( Fsipgautirael 4a)r.rangements of Cys residues among structures and related metal–
sulfur clusters were retrieved (Figure 4).

Figure 4. Metal–thiolate cluster analysis and structural similarities between PlMT7, PlMT8 and
Figure4.Metal–thiolateclusteranalysisandstructuralsimilaritiesbetweenPlMT7,PlMT8andSpMTA.
SpMTA. (A) Cluster connectivities between the cysteine thiolate groups and the metal ions in the α-
(A) Cluster connectivities between the cysteine thiolate groups and the metal ions in the ↵- and
and β-domains of the sea urchin MTs. Cys residues are boxed in yellow, while the metal ions are
�-domainsoftheseaurchinMTs.Cysresiduesareboxedinyellow,whilethemetalionsarespecifiedby
specified by roman numerals; (B) Superposition of the 3D structures of PlMT7 and PlMT8 with
romannumerals;(B)Superpositionofthe3DstructuresofPlMT7andPlMT8withSpMTA.Proteinsare
SpMTA. Proteins are in ribbon representation. The Cys residues are in stick representation; a Gln
inribbonrepresentation. TheCysresiduesareinstickrepresentation;aGlnresidueisalsoshown.
TrehseidPu.lei visid aulssop rsohtoewinns.a Trehein Pi.v loivriyd,utsh per↵o-tdeionms aairne oinf SivpoMryT,A thies sαh-dowomnaiinnc oyfa SnpaMndTAth ies �sh-doowmna iinn cisyainn
and the β-domain is in violet. Superposition was created and rendered using Chimera package.
violet.SuperpositionwascreatedandrenderedusingChimerapackage.
It should be noted that computational prediction of the metal binding sites in P. lividus as well
ItshouldbenotedthatcomputationalpredictionofthemetalbindingsitesinP.lividusaswellas
as in S. purpuratus MTs suggests alternative thiolate clusters, composed by Cys encompassing
inS.purpuratusMTssuggestsalternativethiolateclusters,composedbyCysencompassingresidues
residues from both the N- and C-domain. Thus, it could be hypothesised that a wide range of
fromboththeN-andC-domain. Thus,itcouldbehypothesisedthatawiderangeofconformational
conformational states and metal interactions between domains could be supported.
statesandmetalinteractionsbetweendomainscouldbesupported.
2.3. Phylogenetic Analysis of Deuterostome MTs
2.3. PhylogeneticAnalysisofDeuterostomeMTs
In order to study metallothionein evolution in deuterostomes, annotated protein sequences were
Inordertostudymetallothioneinevolutionindeuterostomes,annotatedproteinsequenceswere
retrieved from databases. Moreover, when not already annotated, MT sequences were searched by
retrievedfromdatabases. Moreover,whennotalreadyannotated,MTsequencesweresearchedby
similarity in transcriptome databases (Table 1). All available transcriptomes of Echinodermata
similarityintranscriptomedatabases(Table1). AllavailabletranscriptomesofEchinodermataspecies
species were considered, including Asteroidea (sea stars), Ophiuroidea (brittle stars), and
were considered, including Asteroidea (sea stars), Ophiuroidea (brittle stars), and Holothuroidea
Holothuroidea (sea cucumbers). Moreover, for understanding and gaining insight into the
evolutionary trends of the MTs, phylogenetically interesting species belonging to non-vertebrate
chordates as amphioxus and ascidians were considered [28–30]. Additionally, jawless vertebrates
Int.J.Mol.Sci.2017,18,812 6of26
(seacucumbers). Moreover,forunderstandingandgaininginsightintotheevolutionarytrendsof
theMTs,phylogeneticallyinterestingspeciesbelongingtonon-vertebratechordatesasamphioxus
and ascidians were considered [28–30]. Additionally, jawless vertebrates (agnates/Cyclostomata),
representativesofanancientvertebratelineage,andrelativelyearlydivergingteleostfishes,likeeels
andcatfisheswereselected. Alsotwowell-studiedfishes,theJapanesetigerpufferandthezebra-fish,
andahumanMTwerechosen[31,32]. Unfortunately, noacornworm(Hemichordata)norsealily
(Crinoidea)MTsequenceswerefound. TheMSAbetweenMTsfromthesedifferentclassesisshown
in Figure 5. The alignment was also used to construct the phylogenetic tree shown in Figure 6.
Asanoutgroup,anMTfromtheciliateprotozoaTetrahymenapyriformiswasselected.TetrahymenaMTs
constituteanexcellentexampleofanMTsubfamilywellstudiedintermsofmoleculargeneticsand
proteinlevels[33–35]. Thissubfamilyisconsiderablydivergentfromthedeuterostomeparadigm,
soitisunambiguouslyoutsidethecladeofinterestinthisphylogeneticstudy. Nevertheless,theycan
be successfully aligned to sequences from the ingroup. Since the three MT isoforms identified in
T. pyriformis are longer than deuterostome ones [36–38], we selected the shortest one (MT-2) as
theoutgroup.
The phylogenetic analysis of echinoderm MT sequences suggested that many events of gene
duplicationhaveoccurredindependentlyindifferentspecies.Foreachspeciesthesequencedivergence
isverydifferent,suggestingthatgeneduplicationoccurredafterspeciationsandatdifferenttimes.
MTs of echinoderms exhibit high sequence heterogeneity, both among them and in relation to the
vertebratepeptides,andPlMTsappearthemostdivergent. MTsofEucidaristribuloides,arepresentative
cidaroid, branch apart from other sea urchin sequences, confirming cidaroids are the basal group
within the class [39]. Echinarachnius parma, a representative clypeasteroida, results in the cidaroid
sisterbranch.
Even though the inter-relationships of the classic deuterostome phyla appear to be resolved,
the intra-relationships within each of the phyla have been troublesome and not completely
concluded[40].TheanalysisofthedifferentclassesofEchinodermataMTssuggeststhatophiuroidMTs
weremorecloselyrelatedtoasteroidthantotheechinozoanMTsand,inturn,totheholoturianMTs.
Asalreadyreported,theMTsequencesoftunicatesandamphioxusshowalowidentitywith
otherdeuterostomeMTs,notbeingdirectlyalignabletotheisoformsofotherclasses[28,30]. Thus,
BranchiostomaMTsappearinaseparatetreebranchwithrespecttothetwotunicatesandgroupwith
theechinodermisoformsatagreaterdistanceofthevertebratecounterparts. TheseMTsarethemost
similartoMTsofmolluscs(datanotshown). SeasquirtMTsaretheshortestMTssofaridentified
indeuterostomesandareclearlyseparatedfromotherChordataandEchinodermataMTs. Finally,
theMTsoflampreys, representativesofanancientvertebratelineagethatdivergedfromourown
~500Myr,asexpected,appearthenearestrelatedtoothervertebrataMTs.
InordertoanalysealsoMTgenestructureevolution,newBLASTsearchesingenomicdatabases
were performed using MT mRNA sequences as queries (Table 1). Genomic sequences of already
annotatedMTgeneswerealsoretrievedfromdatabases. Novelannotatedgenesfromselectedgenome
draftsequenceswerecomparedtoalreadyknownMTgenes.
Int.J.Mol.Sci.2017,18,812 7of26
Table1.Species,protein,transcriptandgenesequenceIDscollectedforphylogeneticalandgenestructuralanalyses.
Taxonomy Species Name IDProtein/Reference IDmRNA IDGene/Scaffold#
EchinoideaEchinoida Paracentrotuslividus PlMT4/8 Ragusaetal.,2013 §
EchinoideaEchinoida Sterechinusneumayeri SnMT P55953
SpMTA P04734.2 SPU_017989.3a Scaffold1168v3.1
Strongylocentrotus
EchinoideaEchinoida SpMTB2 Q27287 SPU_017134.3c Scaffold1906v3.1
purpuratus
SpMTB1 SPU_001866.3a Scaffold249v3.1
HtMT1 comp104953_c1_seq2⌃
EchinoideaEchinoida Heliocidaristuberculata §
HtMT2 comp104953_c1_seq1⌃
SgMTA Q26497
EchinoideaTemnopleuroida Sphaerechinusgranularis
SgMTB Q26496
EchinoideaTemnopleuroida Lytechinuspictus LpMT1 O02033
AlMT1 c31462_g1_i3¶
EchinoideaArbacoida Arbacialixula §
AlMT2 c31386_g2_i2¶
Echinoidea
Echinarachniusparma EpMT § GAVF01002251
Clypeasteroida
EtMT1 JI315060 §JZLH010301553
EchinoideaCidaroida Eucidaristribuloides EtMT2 § GAZP01041405
EtMT3 JI295076
Asteroidea Asteriasforbesi AfMT § GAUS01062044.1
Asteroidea Asteriasrubens ArMT § GAUU01048766.1
Asteroidea Asteriasamurensis AamMT § GAVL01015559.1
Asteroidea Leptasteriassp.AR-2014 LsMT § GAVC01041863
OeMT1 GAUQ01108229
Ophiuroidea Ophiocomaechinata OeMT2 § GAUQ01027563
OeMT3 GAUQ01073893
HolothuroideaAspidochirotida Apostichopusjaponicus AjMT § GH551565 §MODV01037111+MODV01043468
§Scaffold4005KN886207.1+
HolothuroideaAspidochirotida Parasthicopusparvimensis PpMT § Locus_1_Transcript_31588/53328
Scaffold3285KN885487.1
HolothuroideaAspidochirotida Parastichopuscalifornicus PcMT1 § GAVO01014408.1
HolothuroideaDendrochirotida Sclerodactylabriareus SbMT § GAUT01018048.1
Int.J.Mol.Sci.2017,18,812 8of26
Table1.Cont.
Taxonomy Species Name IDProtein/Reference IDmRNA IDGene/Scaffold#
Crinoidea -
Hemichordata -
UrochordataEnterogona Cionaintestinalis CiMT1 ACN32211.2Franchietal.,2010 BW491384FJ217357 Scaffold186(JGIdatabase)
UrochordataEnterogona Ascidiasydneiensissamea AsMT Yamaguchietal.,2004 Asy-sig-715andAsy-sig-997
HcMTA AY314949
UrochordataStolidobranchia Herdmaniacurvata §
HcMTb AY314939.1
UrochordataStolidobranchia Herdmaniamomus HmMT § EL733027.1
BfMT1 BW764364
CephalochordataAmphioxiformes Branchiostomafloridae Guirolaetal.,2012 Guirolaetal.,2012
BfMT2_S FE561990.1
CephalochordataAmphioxiformes Branchiostomabelcheri BbMT20 XP_019631158.1 XM_019775599.1 NW_017803933(AYSS01018500)
§APJL01076593+APJL01076594+
CraniataCyclostomata Lethenteroncamtschaticum LcMT § DC612982.1
APJL01076595
CraniataCyclostomata Petromyzonmarinus PmMT § CO548937.1
CraniataGnathostomataElopomorpha Anguillaanguilla AaMT ABF50549.1 DQ493910.1 §AZBK01727549
NM_001200077.1orAF087935
CraniataGnathostomataOtomorpha Ictaluruspunctatus IpMT O93571 NC_030419
andJT349175.1
CraniataGnathostomataOtomorpha Clariasmacrocephalus CmMT AGC79138.1 JX312865.1
DrMTBl ENSDARP00000131449
ENSDART00000170342 ENSDARG00000102051
CraniataGnathostomataOtomorpha Daniorerio (like) (CAA65933.1)
ENSDARP00000061006
DrMT2 ENSDART00000061007 ENSDARG00000041623
(AAH49475.1)
CraniataGnathostomata
Takifugurubripes TrMT2 ENSTRUP00000022394 ENSTRUT00000022487 ENSTRUG00000008907.1
Euteleosteo-morpha
CraniataGnathostomataPrimates Homosapiens HsMT1A NP_005937.2 NM_005946 GeneID:4489
Scaffold#:Scaffoldnumber.§Sequencesidentifiedinthiswork.⌃HeliocidarisT_Trinitydatabase.¶AvailableinEchinobase.
Int.J.Mol.Sci.2017,18,812 9of26
Int. J. Mol. Sci. 2017, 18, 812 9 of 25

Figure 5. MSA of MT protein sequences. Conservation score higher than 0.5 is highlighted in yellow.
Figure 5. MSA of MT protein sequences. Conservation score higher than 0.5 is highlighted in
Unaligned cysteines are in orange. Cysteines aligned in all sequences are highlighted in red. Dots
yellow. Unaligned cysteines are in orange. Cysteines aligned in all sequences are highlighted in
denote gaps. Species acronyms, taxonomy and sequence IDs are indicated in Table 1.
red.Dotsdenotegaps.Speciesacronyms,taxonomyandsequenceIDsareindicatedinTable1.
Int.J.Mol.Sci.2017,18,812 10of26
Int. J. Mol. Sci. 2017, 18, 812 10 of 25

FigurFei g6u. rAem6.inoA maciindo seacqiudesnecqeu-benacsee-db anseeidghnbeoiguhrb-joouinr-ijnoign itnrege.t rSepe.eciSepse acciersonacyrmonsy amnsd asnedquseenqcuee nIDces are
IDs are indicated in Table 1. The values at nodes indicate bootstrap support greater than 50%.
indicated in Table 1. The values at nodes indicate bootstrap support greater than 50%. Tetrahymena
Tetrahymenapyriformiswasusedasoutgrouptorootthetree.Branchlengthsaredrawntoascaleof
pyriformis was used as outgroup to root the tree. Branch lengths are drawn to a scale of amino acid
aminoacidsubstitutionspersite.
substitutions per site.
Forthefirsttime,MTgenesoftheslatepencilurchinoftheCidaroidaorder(E.tribuloides),twosea
For the first time, MT genes of the slate pencil urchin of the Cidaroida order (E. tribuloides), two
cucumbers(ApostichopusjaponicusandParasthicopusparvimensis),alamprey(Lethenteroncamtschaticum)
sea cucumbers (Apostichopus japonicus and Parasthicopus parvimensis), a lamprey (Lethenteron
andaneel(Anguillaanguilla)werecharacterized. GenestructuresaredepictedinFigure7andshow
camtschaticum) and an eel (Anguilla anguilla) were characterized. Gene structures are depicted in
clearlythehighvariabilityofintronlengths,thelongestbeingtheCidaroidaE.tribuloidesgeneand
Figure 7 and show clearly the high variability of intron lengths, the longest being the Cidaroida E.
theshortestthefishgenes. Alltheintronsthatinterruptthecodingregionsoftheselectedgenesare
tribuloides gene and the shortest the fish genes. All the introns that interrupt the coding regions of the
inphase-1andallthecodingregionswiththeexclusionoftheascidianone[28]areinterruptedby
selecttewdo ginetnroesn sa.rIen tienr epshtiansgely-1, tahnedla malpl rtehye gceondeincogn rtaeignisonalss owtihteh itnhtreo enxicnluthseio3n UoTf Rthleik aeseccihdiinaond oenrme [28]
0
are inatnedrrauspcitdeida nbyo ntews.oA instarlorenasd. yInrteeproerstteind,gllayn, ctehleet l(aBmrapnrcehyio sgteonmea cfloornitdaaien)sg eanlseos ttrhuec tiunrterosnar ieni nthseo m3′ eUTR
like wecahyinuondiqeurem,i nadneded aBsfMcidTi1alna ckosntehse. inAtsro nalirnetahdey3 0rUepTRoratnedd,t hleanBcfMelTet2 fi(Brsrtainncthroionsitsomloac atfelodriadtaoen) lygene
struc4tucroedso anrsef rionm sothmeeA wTGay[ 3u0n].iqTuhue,s ,inaldsoeegde nBefMstrTu1ct luarcekssr etflheec tindtirvoenrg ienn ctheein 3c′ oUdTinRg asenqdu ethncee Bs.fMT2 first
intron is located at only 4 codons from the ATG [30]. Thus, also gene structures reflect divergence in
coding sequences.