Computational and functional analysis of biopharmaceutical drugs in zebrafish: Erythropoietin as a test model

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ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Computational

and

functional

analysis

of

biopharmaceutical

drugs

in

zebraﬁsh:

Erythropoietin

as

a

test

model

Michela

Guarienti

a,∗

_,

_Edoardo

_Giacopuzzi

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_,

_Alessandra

_Gianoncelli

a

_,

_Sandra

_Sigala

a

_,

Pierfranco

Spano

a

_,

_Sergio

_Pecorelli

a,b

_,

_Luca

_Pani

b

_,

_Maurizio

_Memo

a

a_Department_of_Molecular_and_{Translational}_Medicine,_University_of_Brescia,_Viale_Europa_11,₂₅₁₂₃_Brescia,_Italy b_Agenzia_Italiana_del_Farmaco,_Via_del_Tritone_181,₀₀₁₈₇_Roma,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14May2015

Receivedinrevisedform5August2015 Accepted4September2015

Availableonline8September2015 Keywords: Zebraﬁsh Erythropoietin 3Dmodeling Biosimilars Functionalanalysis Inﬂammatoryresponse

a

b

s

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Thezebraﬁsh(Daniorerio)isaverypopularvertebratemodelsystem,especiallyembryosrepresenta valuabletoolforinvivopharmacologicalassays.Thisismainlyduetothezebraﬁshadvantageswhen comparedtootheranimalmodels.

Erythropoietinisaglycoproteinhormonethatactsprincipallyonerythroidprogenitors,stimulating theirsurvival,proliferationanddifferentiation.Recombinanthumanerythropoietin(rhEPO)hasbeen widelyusedinmedicinetotreatanemiaanditisoneofthebest-sellingbiotherapeuticsworldwide. Therecombinantmolecule,industriallyproducedinCHOcells,hasthesameaminoacidsequenceof endogenoushumanerythropoietin,butdiffersintheglycosylationpattern.Thismayinfluenceefficacy andsafety,particularlyimmunogenicity,ofthefinalproduct.

Weemployedthezebraﬁshembryoasavertebrateanimalmodeltoperforminvivopharmacological assays.WeconductedafunctionalanalysisofrhEPOalphaEprex®andtwobiosimilars,theerythropoietin alphaBinocrit®andzetaRetacrit®.

Byinsilicoanalysisand3Dmodelingweprovedtheinteractionbetweenrecombinanthuman erythro-poietinandzebrafishendogenouserythropoietinreceptor.Thenwetreatedzebrafishembryoswiththe3 rhEPOsandweinvestigatedtheireffectonerythrocytesproductionwithdifferentassays.Byrealtime-PCR weobservedtherelativeupregulationofgata1(2.4±0.3fold),embryonic␣-Hb(1.9±0.2fold)and␤-Hb (1.6±0.1fold)transcripts.AsignificantincreaseinStat5phosphorylationwasalsoassessedinembryos treatedwithrhEPOswhencomparedwiththenegativecontrols.Liveimagingintg(kdrl:EGFP; gata1:ds-red)embryos,o-dianisidinepositiveareaquantificationandcyanomethemoglobincontentquantification revealeda1.8±0.3foldincreaseoferythrocytesamountinembryostreatedwithrhEPOswhencompared withthenegativecontrols.Finally,weverifiedthatrecombinanthumanerythropoietinsdidnotcause anyinflammatoryresponseinthetreatedembryos.

Ourdatashowedthatzebraﬁshembryocanbeavaluabletooltostudyinvivoeffectsofcomplex pharmacologicalcompounds,suchasrecombinanthumanglycoproteins,allowingtoperformfastand reproduciblepharmacologicalassayswithexcellentresults.

Abbreviations: hpf,hourspostfertilization;dpf,dayspostfertilization;CHO,Chinesehamsterovary;EPO,erythropoietin;EPOR,erythropoietinreceptor;rhEPO, recombinanthumanerythropoietin;hepo,humanerythropoietingene;hEPO,humanerythropoietinprotein;hEPOR,humanerythropoietinreceptorgene;hEPOR,human erythropoietinreceptorprotein;zepo,zebrafisherythropoietingene;zEpo,zebrafisherythropoietinprotein;zepor,zebrafisherythropoietinreceptorgene;zEpor,zebrafish erythropoietinreceptorprotein;WISH,wholemountinsituhybridization.

∗ Correspondingauthor.Fax:+390303717529.

E-mailaddresses:[email protected](M.Guarienti),[email protected](E.Giacopuzzi),[email protected](A.Gianoncelli),

[email protected](S.Sigala),[email protected](P.Spano),[email protected],[email protected](S.Pecorelli),[email protected](L.Pani),

[email protected](M.Memo).

http://dx.doi.org/10.1016/j.phrs.2015.09.004

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1. Introduction

Thezebrafish(Daniorerio)iswidelyusedasanexperimental vertebrateanimalmodelinmanyfieldsofscientificresearch.For instance,becauseofeggstransparency,rapidexuterodevelopment andeasyaccesstoexperimentalmanipulation,zebrafishembryos represent a valuable tool for in vivo pharmacological assays, offeringseveraladvantageswhencomparedtootheranimal mod-els.Strongcorrelationshave beenobservedbetweentheeffects ofmanycompoundstestedbothonzebrafishandhumans. More-over,drugadministrationtoembryosissimpleandfast,andalarge numberofindividualscanbeanalyzedatthesametime[1–3].

Thezebrafishisalsoaveryusefulanimalmodeltostudyinvivo vertebratehematopoiesis[4].Thegenesandthemolecular path-waysinvolvedinhematopoiesisareverywellconservedbetween zebrafishandhighervertebrates.Moreover,morphologyand func-tionofzebrafishbloodcellsarecomparabletotheirmammalian counterparts[5,6].

The ﬁrst myeloid progenitors mature into primitive macrophagesandgranulocytes,whichhavetheabilitytomigrate towardsthesiteofawoundandtophagocytoseforeignparticles by 2 days postfertilization (dpf). Proerythroblasts are theﬁrst bloodcellenteringthecirculationaround24hpfandmatureinto primitiveerythrocytes,thataretheonlycirculatingredbloodcells till4dpf[5,7].

Zebraﬁsherythrocytesshowthetypicalellipticalshapeand,as alltheothernonmammalianvertebrates,theyretaintheirnucleus alsointhematureadultstage.Erythropoiesisisﬁnelyregulatedby differentmolecularmechanisms,suchastranscriptionfactors(i.e., scl,lmoandgatafamily)oractivationofsignalingpathways,the mostimportantofwhichistheerythropoietinpathway[8].

Erythropoietin (EPO) is a glycoprotein hormone that acts principallyon erythroid progenitors, stimulating their survival, proliferation and differentiation. In mammals,EPO is produced by fetal hepatocytes during development and mainly by per-itubularinterstitialﬁbroblastsinadultkidney.EPObindingtoits homodimericreceptor(EPOR)onprogenitorssurface,causesa con-formational changein EPOR intracellulardomain. Thisleadsto JAK2transphosphorylationand EPORactivation,whichactivates thedownstreamsignalingcascadeviaSTAT5,PI3K/AKTandMAPK pathways[9].

Humanerythropoietingene(hEPO)wasclonedand character-izedfor theﬁrsttime in1985[10,11].Since then,recombinant DNAtechnologyallowedtheproductionofrecombinant human erythropoietin(rhEPO)usingmammalianChinesehamsterovary (CHO)cellsasexpressionsystem.rhEPOhasbeenwidelyusedto treatpatientswithchronicanemiacausedbyrenalfailure,butalso chemotherapy-associatedanemiaincancerpatients,severe ane-miaduetoantiviraltherapy inAIDSpatientsandperioperative anemiaaftersurgery[12].

In 2007, two decadesafter human erythropoietin synthesis, zebrafisherythropoietinanderythropoietinreceptorgenes(zepo andzepor,respectively)wereclonedandbiochemically character-ized[13,14].Ithasbeenshownthat,despitethelowidentityscore betweenhumanandzebrafisherythropoietinproteins(hEPOand zEpo,respectively),themostimportantresiduesaremaintained amongthem.Aswell,proteinsequencesofhumanandzebrafish erythropoietinreceptors(hEPORandzEpor,respectively)sharelow identityscore,butverywellconservedfunctionaldomains[15].The biologicalfunctionofbothzEpoandzEporseemstobemaintained: downregulationofzepoorzeporgenescausesadramaticdecrease ofhemoglobin,whileoverexpressionofzepoleadstoanincreasein circulatingerythroidcells[15].

Duetotheseﬁndings,weemployedthezebraﬁshembryoas avertebrateanimalmodeltostudytheeffectsof3commercially availablerhEPOsinvivo.Inthepresentpaperweperformeda

func-tionalanalysisofEprex®anditsbiosimilarsBinocrit®andRetacrit®

[16].Eprex®wastheﬁrstpatentedrhEPOapprovedbyFDAin1989, whiletheerythropoietinalphaBinocrit®andzetaRetacrit®were authorizedasEprex® biosimilarsin2007.Becauseofthesimilar aminoacidsequenceandtheglycosylationpattern,the erythropoi-etinzetaRetacrit®isconsideredhomologousoftheerythropoietins alpha[17].Bioequivalenceoferythropoietinalphaandzetawas demonstratedbypharmacokineticstudies[18].Thebiosimilarity betweenthe3differentrhEPOproteinswasalsodemonstratedin ourlaboratorybyaproteomicapproach(submittedmanuscript).

Inthepresentstudywefirstperformedacomputationalanalysis topredictifrhEPOcouldinteractwithzEpor.Wetreatedzebrafish embryoswiththe3 rhEPOsandwe investigatedtheireffecton erythrocytesproduction,analyzingchangesbothinqualityandin quantity.Moreover,weexaminedwhetherthepresenceofthese exogenouscompoundscouldcauseaninflammatoryresponsein thetreatedembryos.Inthispaper,wefirstlyshowthatzebrafish embryo canbeagood animal modeltostudyinvivoeffects of complexpharmacologicalcompounds.

2. Materialandmethods

2.1. Insilicoanalysis

TheaminoacidsequenceofrhEPOpublishedontheEuropean Pharmacopoeia8th edition(http://online6.edqm.eu/ep805/)was usedtoBLASTsearchthehumanandzebraﬁshEnsemblgenome sequencedatabaseathttp://www.ensembl.orgThedatabase sup-pliesthemoreupdatedgenomeassembly,whichisGRCh38(Db version78.38)forhuman,andZv9(Dbversion78.9)forzebraﬁsh

[19].The“tblastn”andthe“blastp”algorithmsallowedusto iden-tifythehumanendogenouserythropoietintranscriptandprotein sequences,respectively.Threezebrafishsplicevariantsofthesame erythropoietin gene were found, and each of these transcripts encodedforaprotein.ThehEPOproteininformationcollectedin theUniProtdatabase(http://www.uniprot.org)wereusedtoobtain hEPORentry,inthe“ProteinInteraction”section[20].Ensemblfull lengthsequencesofhumanerythropoietinreceptortranscriptand proteinwerededucedandusedtoBLASTsearchthezebrafishZv9 Ensemblgenomeassembly. Onefulllengthzebrafishtranscript, encodingforoneerythropoietinreceptorprotein,wasidentified.

Acomparativeanalysisofgeneorganizationbetweenhuman andzebraﬁsherythropoietinandEPOreceptorgeneswascarried out,employinginformationsuppliedbytheEnsembldatabase[19]. Synteny analysiswasperformedusingtheSynteny database (http://syntenydb.uoregon.edu/syntenydb/) and the Genomi-cus genome browser (http://www.genomicus.biologie.ens.fr/ genomicus-78.01/cgi-bin/search.pl)[21,22].

TheproteinsequencesofrhEPO,endogenoushEPOand zEpo were employed to perform multiple sequence alignment by ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/)

[23].ThesameanalysiswascarriedoutusinghEPORand zEpor proteinsequences.

2.2. Homologymodeling

To betterevaluatethe functional similaritybetween human and zebraﬁsh erythropoietin receptors and to assess if rhEPO couldrecognize andbind thezebraﬁsherythropoietin receptor, a 3D model of zEporwas created.The zEporprotein sequence (NP001036799.1)andPSI-BLASTwereusedtoidentifyalistof7 suitabletemplatesfromtheRCSBproteindatabank(http://www. rcsb.org/pdb/home/home.do)[24].

The 1EERcomplex, representing theextracellular portionof hEPOR,wasselectedasbesttemplategivenitssimilaritywithzEpor

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andtheinclusionofthehumanEPOsubstrate.Asinglechain(chain B)fromthistemplatewasusedinI-Tasser(http://zhanglab.ccmb. med.umich.edu/I-TASSER/) [25] to performhomology modeling andgeneratethepredictedstructureofzEporextracellulardomain, identifiedfromresiduesY26toT232.TheN-terminaldomainand thetransmembraneandintracellularregionswereexcluded.The final model of zEpormonomer was then superimposed onthe humanprotein structure to generatea prediction of thezEpor homo-dimerandtoevaluatetheplacementofrelevantresidues involvedindisulfidebondsandcontactsurfacewithhEPO.Protein structurewasvisualizedandrefinedusingPyMOLv1.7.0.

TobetteranalyzethepossibleinteractionbetweenzEpor homo-dimer and hEPO, a complex between the two molecules was predictedusingGRAMM-X[26],withthe3residuesF108,I164and Y207,imposedasobligatecontactaminoacidsinzEpor.Thebest predictedcomplexthatresemblethecorrecthEPOorientationwas thenusedtoevaluatecontactsurfacesandplacementofrelevant residuesinzEpormodel.

ThesurfaceinteractionbetweenzEporandhEPO was evalu-atedusing SCprogram fromCCP4 package [27,28],considering thepredictedcomplex ofhEPO ligandandzEporstructure, and aninteractionspaceof5Å.Thesamecalculationwasperformed onthe1EERstructureconsideringchainBofthehEPORandhEPO molecules.

2.3. Fishmaintenanceandeggcollection

Allembryoswerehandledaccordingtonationaland interna-tionalanimalcareguidelines.CurrentItalianrulesdonotrequire approvalforresearchonzebraﬁshembryos.

Adultzebrafishoftwostrainswereusedforeggproduction:the wildtypeABstrainandthetransgeniclinecaspertg(kdrl:EGFP; gata1:ds-red), the last one a kindgift of Prof. Cotelli (Depart-mentofBiosciences,UniversityofMilan).Fishesweremaintained inthezebrafish facility atUniversity ofBrescia(Department of MolecularandTranslationalMedicine)understandardlaboratory conditionsasdescribed,at28◦Cona14hlight/10hdarkcycle[29]. Fertilizedeggswerecollectedimmediatelyafterspawning, har-vested,washedandplacedinfishwater.Thedevelopingembryos wereraisedin a 28◦C incubator in0.003% 1-phenyl-2-thiourea (Sigma–Aldrich)topreventpigmentation.

2.4. rhEPOadministration

ThreecommerciallyavailablerhEPOs,soldasinjectable solu-tionsinpreﬁlledsyringes(1mlat40,000IU/ml),wereusedasstock solutions.Thefollowingbatchwereemployed:Eprex® (Janssen-Cilag©₎_#DGS5G00,_Binocrit®_(Sandoz©₎_#47021202_and_Retacrit®

(Hospira©₎_#3E366G3.

Eprex®,Binocrit® and Retacrit® stocksolutionswerediluted totheﬁnal concentrationof 48IU/mlin 0.05%phenolred solu-tion (Sigma–Aldrich). The 0.05% phenol red solution without any pharmaceutical compound was used as negative control. Thecompoundswereadministeredintwodifferentwaystothe dechorionatedembryos.For thesubsequenterythrocytes visual-izationandquantiﬁcationexperiments,4nlofeachcompoundwas injectedintothecommoncardinalveinof48hpfembryos[30].To assessprimitivemyelocytesactivation,1nlofeachdilutionwas injectedinto theotic cavity of 72 hpfembryos. Escherichiacoli JM109bacteriain0.05%phenolredsolutionwereusedaspositive control[31].

2.5. RealtimequantitativeRT-PCR(Q-RT-PCR)

TotalRNAwasextractedfromgroupsof30embryosforeach injectedcompoundusingtheRNAeasykit(Qiagen)and1mgwas

transcribed into cDNA, using the M-MLV reverse transcriptase (Promega).Relativegeneexpressionofgata1,␣-eHband␤-eHb[32]

wasevaluatedbyQ-RT-PCRwiththeViiA7RealTimePCRSystem (LifeTechnologies),usingtheSYBRGreenMethod(Bio-Rad). Analy-siswasperformedintriplicate,Ctwascalculatedusing␤-actinas thehousekeepinggene.Theexperimentwasrepeatedtwice,using differentgroupsofembryos.

2.6. Westernblot

Totalembryoproteinextractswereobtainedby homogeniz-inggroupsof10treatedembryosinlysisbuffer(10mMTrispH 7.4,150mMNaCl,5mMEDTA,10%TritonX-100withacocktail ofprotease andphosphataseinhibitors). ForWesternblot anal-ysis 25␮g of protein extracts were loaded per lane on a 10% Mini-PROTEANTGXGel(Bio-Rad)andtransferred toa nitrocel-lulose paper. The membrane was incubated with1:200 rabbit anti-Phospho-Stat5primaryantibody(CellSignaling)overnightat 4◦C,followedby1:1500IRDye800RDor680RDsecondaryantibody (LI-CORBiotechnology) for 1hat RT.Membranes werewashed andreprobedwithanti-Actinprimaryantibody(Sigma–Aldrich). TheOdysseyInfraredImagingSystem(LI-CORBiotechnology)was employedfor protein detection.The Westernblotanalysis was repeated3times.

2.7. Erythrocytesvisualizationandquantiﬁcation

Treatedembryoswereincubatedat28◦Cfor4hafterinjection andthenusedforerythrocytesvisualizationand/orquantiﬁcation. Eachexperimentwasrepeated3times.

Groups of 25 transgenic casper treated embryoswere anes-thetized in tricaine(Sigma–Aldrich) and photographedunder a LeicaMZ16FstereomicroscopeequippedwithGFP3andG fluores-cencefilters,DFC480digitalcameraandLASLeicaImagingsoftware (Leica,Wetzlar,Germany).Redandgreensignalswerequantified byusingImageJ1.45simageanalysissoftware.

Groups of 25 wild type embryos for each injected com-pound were ﬁxed in 4% paraformaldehyde overnight at 4◦C. O-Dianisidinestainingwasperformedasdescribed inliterature todetect hemoglobin in red blood cells [13]. To visualize ery-throcytesdistribution,embryosweremountedinagarose-coated dishesandphotographed.Imageswereusedtoquantify erythro-cytesonselectedregionofinterestbyusingImageJ1.45simage analysissoftware.

Hemoglobinquantiﬁcation ontotalembryo extractwas per-formedbyusinga modiﬁed cyanomethemoglobinmethod[33]. Groups of 20 wild type embryos for each injected compound wereanesthetized in tricaine (Sigma–Aldrich)and sonicated in 1ml of Drabkin solution (Sigma–Aldrich). After centrifugation, absorbance of the supernatant of each sample was measured at540nmand it waslinearlycorrelated withhemoglobin con-tent.

2.8. Macrophagesandgranulocytesquantiﬁcation

Groupsof40treatedembryosforeachcompoundwere incu-bated at 28◦C for 2h after injection and then ﬁxed in 4% paraformaldehydeinPBSovernightat4◦C.Whole-mountinsitu hybridization(WISH)wasperformedaccordingtoThisseprotocol

[34].pu1,lplastinandmpxwereusedasprobestodetectleukocyte precursors,macrophages and neutrophils [7,35].Embryos were mountedin agarose-coateddishesand imagesweretaken with aLeicaMZ16F stereomicroscopeequippedwithDFC480digital cameraandLASLeicaImagingsoftware(Leica,Wetzlar,Germany).

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LeucocytesquantiﬁcationwasperformedusingImageJ1.45simage analysissoftware.Theexperimentwasrepeated3times.

2.9. Statisticalanalysis

Quantiﬁcationsareexpressedasmean±standarddeviationof independent experiments. Statistical analysiswere made using GraphPadPrism6.01version(GraphPadSoftware).Analysisof vari-ance(one-wayANOVA)followedbyDunnett’stestwasperformed toevaluatesigniﬁcantdifferencesbetweenthegroupsofdata(p value<0.05).

3. Resultsanddiscussion

In ordertoevaluateifzebrafish couldbeusedasan animal modelforrhEPOfunctionalanalysis,wefirstassessedthe simi-laritybetweenhumanandzebrafisherythropoietinreceptorsby computational analysis and 3D modeling techniques. Then we investigated therhEPOseffects onerythrocytes production and theirinflammatoryresponse.

3.1. Insilicoanalysisandhomologymodeling

InordertoidentifythehEPOgeneorthologinzebraﬁsh,the165 aminoacidssequenceofrhEPOwasusedasqueryinaBLASTsearch performed against the human and zebraﬁsh Ensembl genome sequence database at http://www.ensembl.org [19]. The rhEPO sequencefullymatchedwiththeaminoacids28-192ofthehuman endogenousimmatureerythropoietinproteinENSP00000252723, corresponding to the RefSeq: NP000790.2 on the Reference SequencepeptidedatabaseatNCBI(http://www.ncbi.nlm.nih.gov, RefSeqRelease69)[36].ThehEPOproteinisencodedbythe1330bp transcriptENST00000252723(RefSeq:NM000799.2),whichisthe productofthehEPOgeneENSG00000130427 locatedonhuman chromosome7.

In zebraﬁsh, accordingtoliterature, we found3 splice vari-ants of the same zepo gene (ENSDARG00000055163), located on zebraﬁsh chromosome 7 [15]. The 3 zepo transcripts on the Ensembl database are called respectively epo-001 (ENS-DART00000111066), epo-002 (ENSDART00000020288) and epo-201(ENSDART00000077483).TheycorrespondtotheNCBIzepo isoformsL2(RefSeq:NM001038009),L1(RefSeq:NM001115128) andS(RefSeq:NM001115127),respectively.

Bothhumanandzebraﬁshgeneshaveaverysimilar organiza-tion,consistingof5codingexonsand4introns.Theshortestzepo isoform201(orSisoform),withonlyfourcodingexons,differsfrom theotherones.

Itiswellestablishedthataconservedco-localizationofgene clustersamongdifferentspeciesoftencorrespondtoaconserved proteinfunction[37].Asyntenyanalysiswasperformedbetween thehumanandzebraﬁshchromosome7,inthegenomicregions thatcontaintheerythropoietingene(Fig.S1AandB).Byusingthe Genomicusgenomebrowser(http://www.genomicus.biologie.ens. fr/genomicus-78.01/cgi-bin/search.pl) [22] we found 5 ortholog genesintheerythropoietinsyntenicregion,twoofwhich main-tained the same orientation and 3 orientated in the opposite direction(Fig.S1A).TheanalysiswasrepeatedbyusingtheSynteny database(http://syntenydb.uoregon.edu/syntenydb/)[21],which allowedtoevaluateamoreextendedgenomicregion.Byanalyzing a100-genewindow,3moreorthologgenesassociatedwiththe ery-thropoietingenewerehighlighted,andtheymaintainedthesame orientationonbothchromosomes(Fig.S1B).

TheaminoacidsequencesofhEPOandzEpowereemployed to perform ClustalW multiple sequence alignment [23]. The 3 zebraﬁshsplicevariantsofthezepogene encodefor 3proteins,

thatarenamedrespectivelyEpo-L1(RefSeq:NP001108600), Epo-L2 (RefSeq:NP001033098) andEpo-S (RefSeq:NP001108599). The3isoformsareidentical,exceptforonlyfewaminoacidsin theN-terminal signalpeptide(Fig.S1C),thatiscleavedinthe3 matureforms.TheL1isoformwasusedtoperformprotein align-ment,becauseitisthelongestisoformandthemostabundant[15]. ThehEPOaminoacidsequencecovered99%ofzEposequence,with 34.55%identityand66%similarity(Fig.S1D).Byusingthe infor-mationcollectedintheUniProtdatabase(http://www.uniprot.org)

[20]weidentifiedinthehumanhEPOsequence(UniProt:P01588) themostimportant amino acids,responsiblefor erythropoietin post-translationalmodifications(Fig.S1D).The4cysteineCys34, Cys56,Cys60and Cys188wereconservedin thezEposequence (inposition33,55,59and181,respectively).Theseaminoacids areessentialtoformtwodisulfidebond,necessaryforthecorrect proteinfoldingandfunction.Moreover,theO-glycosylationsitein position153and2outof3N-glycosylationsitesinposition65and 110wereconservedinthezebrafish zEposequence(inposition 144, 64and 104, respectively).Theglycosylationof erythropoi-etinisimportantforitscorrectfunctionandhalflife[38,39]and theseconservedresiduessuggestfunctionalconservationbetween humanandzebrafisherythropoietin.

TheinformationsuppliedbytheUniProtdatabase(http://www. uniprot.org)[20] allowedustoidentify,in the“Protein Interac-tion”section,thehumanfulllengthhEPORentry,UniProt:P19235. This was used tosearch theEnsembl databasefor hEPOR 508 amino acids full length protein sequence (ENSP00000222139)

[19]. The hEPOR protein is encoded by the 2411bp tran-script ENST00000222139, which is the product of the hEPOR geneENSG00000187266locatedonhumanchromosome19.The Ensembl human erythropoietin receptor protein and transcript sequencescorrespondtotheNCBIdatabaseRefSeq:NP000112and RefSeq:NM000121,respectively[36].

ThehEPORsequencewasusedtoBLASTsearchthezebrafish Zv9 Ensembl genome assembly [19]. We identified one full length zebrafish transcript ENSDART00000064033 (RefSeq: NM001043334) which is the product of zepor gene ENS-DARG00000043609, located on zebrafish chromosome 3. The 1656bpzebrafishtranscriptencodedforthe509aazEporprotein ENSDARP00000064032(RefSeq:NP001036799).

Thegeneorganizationofbothhumanandzebrafish erythropoi-etinreceptorisverysimilar,constitutedbyeightcodingexonsand sevenintrons.Thegenomicregionsthatcontaintheerythropoietin receptorgeneonhumanchromosome19andzebrafish chromo-some3 showedahighdegreeof synteny (Fig.S2Aand B).The Genomicusgenomebrowserunderlinedtenorthologgenesinthe syntenicregion(Fig.S2A),whilebyusinga100-genewindowin theSyntenydatabasewefoundninemoreorthologgenes asso-ciatedwiththeerythropoietinreceptorgeneonbothhumanand zebrafishchromosomes(Fig.S2B)[21,22].

A ClustalW multiple sequence alignment was performedby using theamino acidsequences of hEPOR and zEporas inputs (Fig. S2C) [23]. The human protein covered 97% of the whole zebraﬁshsequence,withanidentityscoreof27.11%andan over-allsimilarityof62%.Themostimportantresiduesandmotifsof thehumanhEPORsequence,annotatedintheUniProtdatabase, wereconserved alsoin thezebraﬁsh zEporsequence (Fig. S2C)

[20]. Intheextracellular regionthefourcysteine Cys52,Cys62, Cys91 and Cys107,necessary toformtwo disulﬁdebond, were conserved(respectivelyCys44,Cys54,Cys83andCys100inzEpor). TheWSXWSmotif,essentialforthecorrectfoldingandfunction oftheerythropoietinreceptor,waspresentinbothproteins(aa 233-237inhEPOR,aa221-225inzEpor).Moreover,the3residues responsiblefortheligandbinding,Phe117,Met174and Phe229 in hEPOR,werealso conservedin thezEporsequence (Phe108, Ile164 and Tyr217). Thetransmembrane domainhad two

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con-Fig.1.Structural3DmodelofzEporextracellulardomain.(A),(B)SuperimpositionofzEpor(inblue)onhEPOR(inred)showing:(A)theresiduesinvolvedindisulﬁdebond formation;(B)theresiduesinvolvedinEPObinding.(C),(D)AnalysisoftheinteractionbetweenzEpor(inblue)andhEPO(ingreen)showing:(C)thecontactbetweenhEPO andthethreezEporresiduesinvolvedinEPObinding;(D)surfacerepresentationoftheinteractionbetweenthetwomolecules(Scscore0.507).Aminoacidresiduesrefers tozEporRefSeq:NP001036799.(Forinterpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

servedleucineresidues(Leu265-266and Leu254-255inhuman andzebraﬁshsequences,respectively),necessaryforthecorrect receptordimerizationandtheactivationofthesignalingcascade. ThecytoplasmicregionofbothproteinscontainedBox1(aa 282-290in hEPOR, aa 271-279 in zEpor) and Box2 (aa 328-336 in hEPOR,aa317-325 inzEpor),essentialdomainsforthe interac-tion and activation of JAK2. Alsothe ITIM motif (aa 452-457), containingtwophospho-Tyrresidues,wasconserved.Threeother phospho-tyrosine (Tyr468, Tyr485 and Tyr489), important for downstreamsignaling,wereconservedinhEPORandzEpor pro-teins.

Tobetter evaluatethefunctional similarity betweenhuman andzebrafisherythropoietinreceptorsandtoassessifrhEPOcould recognizeandbindthezebrafisherythropoietinreceptor,a three-dimensionalmodelofzEporwascreated(Fig.S3).WeusedI-Tasser (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) and the struc-ture of the extracellular portion of human EPOR (PDB: 1EER) togeneratethepredicted structuralmodel of zEpor extracellu-lardomainbyhomologymodeling[25].Superimpositiononthe humanproteinstructurerevealedthatthefourcysteineputatively involved in disulfide bonds in zEpor (Cys44,Cys54, Cys83 and Cys100)wereplacedneareachother,inaconfiguration compati-blewiththebond(Fig.1A).Analysisofthe3conservedresidues involved in EPO binding, namely Phe108, Ile164 and Tyr217, showedthattheywereplacedinaconfigurationresemblingthose ofthehumancorrespondingPhe117,Met174andPhe229, respec-tively(Fig.1B).Moreover,thepredictedcontactsurfaceofzEpor wassimilartothatofhEPOR(Fig.1CandD;Fig.S3)[26].The molec-ulardockingofhEPOwiththepredictedstructureofzEporshowed alsoanacceptablecompatibilityofcontactsurfaces(Scscore0.507), comparedtothescoreoftheoriginalhumancomplex1EER deter-minedbycrystallography(Scscore0.71)[27,28].

3.2. Erythrocytesvisualizationandquantiﬁcation

To perform a functional analysis of rhEPO in vivo we con-ducted several preliminary experiments in order to set the optimal concentration to be used in zebraﬁsh embryos. We dilutedEprex® in 0.05%phenolredsolution(Sigma–Aldrich)to a series of concentrations rangingfrom 2 to 48IU/ml. The dif-ferent Eprex® concentrations were injected into the common cardinal vein of 48 hpf embryos and they were incubated at 28◦C forupto12hafterinjection.Asnegativecontrolembryos wereinjectedwiththe0.05%phenolredsolutionwithoutdrug. Finallyweperformedano-dianisidinestainingtodetectredblood cells [13]. Time-course and concentration-curves demonstrated aconcentration-dependentincreaseintheerythrocytesnumber, thatreacheditsmaximum4hafterinjection(datanotshown). Basedontheresultsobtainedinthesepreliminaryexperiments, weusedEprex®,Binocrit® andRetacrit® attheconcentrationof 48IU/ml.

To examine the in vivo effects of rhEPO administration on zebrafisherythropoiesis,theexpressionof3lineage-specificgenes wasinvestigated byrealtime Q-RT-PCRafter rhEPOtreatment. Theanalysiswasperformedintriplicateandtheexperimentwas repeated twice, using different groups of embryos. Total RNA wasextractedfromgroupsof30embryosinjectedwithEprex®, Binocrit®,Retacrit®ornegativecontrolsandgeneexpressionlevels werenormalizedusing␤-actinasthehousekeepinggene.The rel-ativeexpressionofgata1wassignificantlyupregulatedinembryos treatedwithrhEPOscomparedtothenegativecontrols(2.4±0.3 foldincrease),aswellastherelativeexpressionof␣-eHB(1.87±0.2 foldincrease)and␤-eHb(1.57±0.1foldincrease).Nostatistically significantdifferencesingeneexpressionhavebeenfoundbetween Eprex®anditsbiosimilars(Fig.2).

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Fig.2. Relativegeneexpressionlevelsofgata1,˛-eHBandˇ-eHbinembryosinjected withnegativecontrol(Negctrl),Eprex®,Binocrit®orRetacrit®.Q-RT-PCRwas per-formedusingtotalRNAextractedfromgroupsof30embryosforeachinjected compound.Dataareexpressedasfoldchangeoftargetgeneexpression normal-izedtotheinternalcontrolgene(ˇ-actin).Datawereanalyzedaccordingtothe comparativeCtmethod.Asterisksindicatestatisticallysigniﬁcantfoldchangesof the3targetgenesexpression(p<0.05).Theexperimentwasrepeatedtwice,using differentgroupsofembryos.

Fig.3.EffectsofEprex®,Binocrit®andRetacrit®treatmentonStat5 phosphoryla-tion.Westernblotwasperformedontotalproteinextractsobtainedfromgroups of10treatedembryos.Theanti-Phospho-Stat5primaryantibodyrevealedthree isoformsofphosphorylatedStat5,ataround90,70and18kDarespectively.The anti-actinprimaryantibodywasusedasinternalcontrol.Thewesternblotimage wasrepresentativeof3independentanalysis.

To conﬁrm that rhEPO was functionally active on zebraﬁsh embryos, we investigated the effects of Eprex®, Binocrit® and Retacrit®injectiononJak/Statpathway.Thephosphorylation sta-tusofStat5proteinwasexaminedontotalembryoproteinextracts obtainedfromgroupsof10embryosforeachinjectedcompound. Indeed, rhEPObindingto zEporis expectedto causeJak2 acti-vation, which leadsto Stat5 phosphorylation[9]. Asshown in

Fig.3,therabbitanti-Phospho-Stat5primaryantibodywasableto detectbasallevelsof3differentisoformsofphosphorylatedStat5 (Fig.3,lane1)[40].AftertreatmentwithEprex®,Binocrit® and Retacrit®thephosphorylationofStat5,especiallytheshortest iso-form,wasclearlyupregulatedrespecttotheuntreatednegative controls(Fig.3).Ofnote,tothebestofourknowledge,onlyfew papersstudyingphospho-Stat5ontotalembryoproteinextracts are availablein literature. We will furtherinvestigate whythe shortestisoform ofStat5resultedthemostphosphorylatedone afterrhEPOtreatment.

The three rhEPOs were then injected in groups of 25 tg (kdrl:EGFP; gata1:ds-red) casper embryos, for subsequent live imagingacquisition.Double-channelﬂuorescenceimages(Fig.4A)

revealedthatbloodvessels(expressingEGFP)wereperviousand red blood cells (expressing ds-red) were able to easily circu-lateinthewholeembryo.Imagesacquiredinsingle-redchannel (Fig. 4B) highlighted an increase of fluorescence intensity in embryosinjectedwithEprex®,Binocrit®andRetacrit®compared to the negative controls.This effect was caused by the higher numberofcirculatingerythrocytesinembryostreatedwith rhE-POs.Itcouldbevisualizedbothintheprincipalvessels(i.e.dorsal aortaandposteriorcardinalvein)andinthesmallestblood ves-sels (i.e.intersegmentalvessels andcaudal venousplexus)[30]. Quantificationoftheredandgreenfluorescencesignal,performed using ImageJanalysis software,is reported inFig. 4C and Das a meanof3independentexperiments.Theredsignalintensity, proportionaltothecirculatingerythrocytes,was2.13±0.09fold higherinembryostreatedwithrhEPOscomparedtothenegative controls.Nostatisticallysignificantdifferenceswerehighlighted betweenthethreebiosimilars(Fig.4C).Onthecontrary,thegreen signalintensity,producedbytheendothelialcellsoftheblood ves-sels,remainedconstantinalltheanalyzedembryos(Fig.4D).

Groupsof25wildtypeembryoswereﬁxedin4% paraformalde-hyde after injectionwithnegative controlor Eprex®, Binocrit® and Retacrit®,each groupwasincubatedino-dianisidine stain-ingbufferandthenphotographed(Fig.5A).Hemoglobincatalyzes theoxidationofo-dianisidine,producingadarkred-brownstainin cellscontaininghemoglobin[13].Erythrocyteswerequantiﬁedin thetrunkandinthetailofeachembryobyusingImageJanalysis software.Fig.5Breportstheresultsofthreeindependent experi-ments:thepercentageofo-dianisidinepositiveareaisproportional totheamountofredbloodcellsmeasuredintheregionofinterest. EmbryostreatedwithrhEPOsshoweda1.67±0.06foldincrease oferythrocytescontentwhencomparedtothenegativecontrols. Interestingly,nodifferenceshavebeenfoundbetweenEprex®and itsbiosimilarsalsointheo-dianisidinestainingassay.

These data were confirmed witha quantitative hemoglobin assayontotalembryoextractbyusinga modified cyanomethe-moglobinmethod[33].Accordingtothemanufacturer,Drabkin’s solution(Sigma–Aldrich)reactswithhemoglobincausingits oxi-dationtomethemoglobin.Inthepresenceofpotassiumcyanide, methemoglobinisconvertedtocyanomethemoglobin,which max-imumabsorbancecanbemeasuredat540nm.Groupsof20wild typeembryosinjectedwithnegativecontrolsolutionorEprex®, Binocrit®andRetacrit®wereanesthetizedintricaineandusedfor theDrabkinassay.Fig.5Cshowsthecyanomethemoglobin opti-caldensity(O.D.)at540nmofeachgroupoftreatedembryos,asa meanofthreeindependentexperiments.Thehemoglobincontent wassignificantlyincreased(1.53±0.05 fold)in embryostreated withrhEPOwhencomparedwiththenegativecontrol.

TheresultsshowedthatEprex®,Binocrit®andRetacrit®were abletointeractwithzebrafishepo-receptor,leadingtoanincrease ofhemoglobincontent,proportionaltothenumberofcirculating erythrocytes.WhencomparedamongthemEprex®,Binocrit®and Retacrit®didnotshowanystatisticallysignificantdifferences. 3.3. Macrophagesandgranulocytesquantification

We then investigated whether Eprex® and its biosimilars Binocrit® and Retacrit® were able to activate primitive mono-cytes/macrophages and granulocytes neutrophils to induce an inflammatorystate.ThethreerhEPOssamples,negativeand pos-itive(E.coliJM109bacteria)controlswereinjectedintotheotic capsuleofgroupsof40healthyzebrafishembryosat72hpf.Itis wellknownthatleukocytesarenormallyabsentinthatanatomical region,butexternalstimulicaninducetheirmigrationtothesiteof infection/inflammation[31].Moreover,ithasbeendemonstrated thatatthisstagemonocytes/macrophagesarepresentinhigher numberandhavemorephagocyticactivitythangranulocytes

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neu-Fig.4. Negativecontrol(Negctrl),Eprex®_,_Binocrit®_or_Retacrit®_injected_in₄₈_hpf_tg_(kdrl:EGFP;_{gata1:ds-red)}_casper_embryos_and_photographed_after₄_h._(A)_Double_channel

fluorescence:ingreenbloodvessels,expressingkdrl:EGFPandinredcirculatingerythrocytes,expressinggata1:ds-red.Lateralviews,anteriortotheleft,11.5Xmagnification; (B)singlered-channelfluorescenceshowscirculatingerythrocytes,expressinggata1:ds-red.Lateralviews,anteriortotheleft,4Xmagnification.Quantificationof(C)red fluorescencesignaland(D)greenfluorescencesignal,measuredwithImageJ1.45simageanalysissoftwareonameanof25embryosforeachexperimentalpoint.Asterisk indicatesstatisticallysignificantincreaseofthepositivearea(p<0.05),dataarethemean±S.D.of3independentexperiments.(Forinterpretationofthereferencestocolor inthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

Fig.5. O-dianisidinestainingandDrabkinassayquantiﬁcationof48hpfwildtypeABembryosinjectedwithnegativecontrol(Negctrl),Eprex®_,_Binocrit®_or_Retacrit®_._(A)

Representativeimagesofembryosstainedwitho-dianisidine4haftertreatment.Lateralviews,anteriortotheleft,4Xmagnification;(B)quantificationofo-dianisidine positiveareaintheregionoftrunkandtail,measuredwithImageJ1.45simageanalysissoftwareonameanof25embryosforeachexperimentalpoint.Asteriskindicates statisticallysignificantincreaseofthepositivearea(p<0.05),dataarethemean±S.D.of3measurements;(C)cyanomethemoglobinabsorbancemeasuredat540nm, proportionaltotheamountofredbloodcellsingroupsof20embryos.Asteriskindicatesstatisticallysignificantincreaseofabsorbance(p<0.05),dataarethemean±S.D.of 3measurements.

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Fig.6. Insituhybridizationwithpu1,lplastinandmpxprobestodetectleukocytesprecursors,monocytes/macrophagesandgranulocytesneutrophils,respectively.(A) Representativeimagesof72hpfembryosinjectedintotheoticcapsulewithEprex®,Binocrit®orRetacrit®andpositiveornegativecontrol(PosctrlandNegctrl,respectively). Lateralviews,anteriortotheleft,11.5Xmagnification;(B)quantificationofpu1,lplastinandmpxpositivearea,measuredwithImageJ1.45simageanalysissoftwareona meanof40embryosforeachexperimentalpoint.Asteriskindicatesstatisticallysignificantincreaseofthepositivearea(p<0.05),dataarethemean±S.D.of3measurements.

trophils[31].PrimitiveleukocytesweredetectedbyWISH,using pu1,lplastinandmpxasmolecularprobes.Thetranscription fac-torpu1isessentialforearlyhematopoiesis,inparticularforthe myeloidcelldevelopment,includingbothmacrophagesand gran-ulocytesprecursor.Theactin-bindingproteinencodedbylplastin ispredominatelyexpressedinmonocytes/macrophages,whilethe enzymemyeloperoxidase,encodedbympx,istipicallylocatedin neutrophilsgranules[7,35].

Fig.6reportsleukocytesresponsetotheinjectionintotheotic capsuleofgroupsof40embryos,asthemeanofthree indepen-dentexperiments.Asexpected,theamountofmacrophageswas abouttwicethenumberofneutrophilsintheoticcapsuleandinthe surroundingareaofalltheanalyzedembryos.Bothmacrophages andneutrophilswerestronglyattractedtotheinjectionsitebythe presenceofthepositivecontrolofE.Colibacteria,whilethe neg-ativecontrolhadaverymildeffectonleukocytes,similartothat ofEprex®anditsbiosimilarsBinocrit®andRetacrit®(Fig.6A).The leukocytessignalintensityinpositivecontrolswas2.25foldhigher thannegativecontrolsforpu1probe,2.35foldhigherforlplastin

probeand4.49foldhigherformpxprobe(Fig.6B).Onthe con-trary,thesignalquantificationforallthethreeprobesinembryos treated with rhEPOs was comparable to that of the negative controls(1.01±0.09foldincrease,Fig.6B).Theresultsshowedthat Eprex®,Binocrit®andRetacrit®wereabletoactonEPO-receptor without causing any acute pro-inflammatory effects on the zebrafishembryos.

4. Conclusions

Due to its valuable characteristics, in the last decade the zebraﬁshembryohasbeenwidelyusedasavertebratemodelfor highthroughputscreeningofchemicals.However,morerecently it has been increasingly exploited to test not only chemically synthesized drugs, but also more complex molecules (i.e., bio-pharmaceuticals) [1–3]. In this paper, we used the zebraﬁsh embryotoperforminvivopharmacologicalassays.Weperformed afunctionalanalysisofhumanrecombinanterythropoietinalpha Eprex®anditsbiosimilarsBinocrit®andRetacrit®.The

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recombi-nantmolecules,industriallyproducedinCHOcells,havethesame aminoacidsequenceofendogenoushEPObutdifferinthe glycosy-lationpattern.Thismayinfluenceefficacyandsafety,particularly immunogenicity,ofthefinalproduct[38,39].

Dataobtainedbycomputationalanalysisand3Dmodeling tech-niquesstronglysuggested thatrhEPOcouldrecognizeand bind thezebraﬁsherythropoietinreceptor.The3DmodelofzEporwas createdbyhomologymodelingstartingfromthestructureofthe extracellularportionofhEPOR.Itconﬁrmedtheresultsobtained withtheClustalWmultiplesequencealignment,inparticularthe structuralconservationofthemostimportantfunctionalresidues. TheanalysisofsurfacesputativelyinvolvedinEPObindingrevealed ahighdegreeofsimilaritybetweenzEporandhEPORstructure, indeedthemoleculardockingofhEPOwiththepredicted struc-tureofzEporshowedacompatibilityofcontactsurfaces(Scscore 0.507).ThesedatasupportedthehypothesisthathEPOcouldbe recognizedandboundbythezEpor.

ThenitwasdemonstratedthatEprex®anditsbiosimilarswere abletoproduceasignificantincreaseofthenumberof circulat-ingerythrocytes in treated embryos.Therelative expression of gata1,␣-eHBand␤-eHbtranscriptswassignificantlyupregulated inembryostreatedwithrhEPOs,aswellasthephosphorylationof Stat5protein.Liveimagingintg(kdrl:EGFP;gata1:ds-red)casper embryosrevealedthatboththeprincipalvesselsandthesmallest bloodvesselswereperviousandtheconcentrationofcirculating erythrocyteswashigherinembryostreatedwithrhEPOrespect tothenegativecontrols.Alsoo-dianisidinepositivearea quantifi-cationandcyanomethemoglobincontentquantificationrevealed asignificantincreaseoferythrocytesamountinembryostreated withrhEPOswhencomparedwiththenegativecontrols.

Finally, this experimental animal model conﬁrmed the lack ofimmunogenicity,required fortheregulatoryapproval of bio-pharmaceuticaldrugs.IndeedembryosinjectedwithEprex®orits biosimilarsshowedanalmostnulleffectonactivationofprimitive monocytes/macrophagesandgranulocytesneutrophils, compara-bletothatofnegativecontrols.

In conclusion, ourdatademonstrated that zebrafish embryo couldrepresentannew,reproducibleandrobustexperimental ver-tebrateanimalmodeltostudytheefficacyandthesafetyofcomplex biotherapeuticsandbiosimilars,suchasrecombinanthuman gly-coproteins.Indeed,usingthezebrafishembryo,wewereableto testinvivoeffectsofrhEPO,intermsofbothbiologicalactivityand safety.Zebrafishembryodemonstratedtobeavaluabletool,that allowstoperformfastand reproduciblepharmacologicalassays withexcellentresults.

Conﬂictofinterest

None.

Acknowledgement

ThisworkwassupportedbyAgenziaItalianadelfarmacoAIFA (ProjectPHARM-Q).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2015.09. 004.

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