同源于hMIP-1α的vMIPα对人趋化因子受体的作用

同源于hMIP-1α的vMIPα对人趋化因子受体的作用Abstract

Humanmacrophageinflammatoryprotein-1alpha(hMIP-1α)andviralmacrophageinflammatoryprotein-alpha(vMIPα)belongtotheCCchemokinefamilyandsharehighsequencehomology.However,theyexhibitdifferentbiologicalactivities,suggestingthattheirinteractionwithchemokinereceptorsmaydiffer.Inthisstudy,weinvestigatedtheeffectofvMIPαonhumanchemokinereceptorsCCR1,CCR2,CCR3,CCR4,andCCR5,whichareknowntobindhMIP-1α.

Usingcalciummobilizationandchemotaxisassays,wefoundthatvMIPαdoesnotactivateanyofthesereceptorsbutinsteadsuppressesthechemotacticresponseofthesereceptorstohMIP-1αinadose-dependentmanner.Furthermore,vMIPαalsoinhibitsthecalciummobilizationinducedbyhMIP-1α.

Usingsite-directedmutagenesis,weidentifiedseveralaminoacidresiduesthatarecriticalfortheinhibitoryeffectofvMIPαonCCR5,includingresiduesinthetransmembranehelicesandextracellularloopsofthereceptor.

OurresultsdemonstratethatvMIPαcanfunctionasachemokinereceptorantagonistformultiplehumanchemokinereceptors,includingthosethatbindhMIP-1α.ThisfindingsuggeststhatvMIPαmayhavepotentialtherapeuticapplicationsfordiseasesassociatedwithchemokinereceptoractivation,suchasinflammationandcancermetastasis.

Introduction

Chemokinesaresmallsecretedproteinsthatplaycrucialrolesintheimmunesystembyregulatingleukocytetraffickingandactivation.Theybindtospecificchemokinereceptorsonthesurfaceofcells,leadingtovariouscellularresponses,includingchemotaxis,calciummobilization,andgeneexpression.Humanmacrophageinflammatoryprotein-1alpha(hMIP-1α)isamemberoftheCCchemokinefamilythatisinvolvedinvariousphysiologicalandpathologicalprocesses,suchasinflammation,viralinfection,andcancermetastasis[1].hMIP-1αbindstomultiplechemokinereceptors,includingCCR1,CCR2,CCR3,CCR4,andCCR5,withdifferentaffinitiesandbiologicalactivities[2,3].

Viralmacrophageinflammatoryprotein-alpha(vMIPα),achemokinehomologencodedbytheKaposi'ssarcoma-associatedherpesvirus(KSHV),shareshighsequencehomologywithhMIP-1αandisknowntobindtoseveralhumanchemokinereceptors,includingCCR3,CCR5,andCXCR4[4,5,6].However,vMIPαexhibitsdifferentbiologicalactivitiesfromhMIP-1α,suchastheinhibitionofT-cellchemotaxis,thesuppressionofangiogenesis,andthepromotionofviralreplication[7,8].TheseobservationssuggestthatvMIPαmayinteractwithchemokinereceptorsdifferentlyfromhMIP-1α.

Inthisstudy,weinvestigatedtheeffectofvMIPαonthechemotaxisandcalciummobilizationofhumanchemokinereceptorsCCR1,CCR2,CCR3,CCR4,andCCR5,whichareknowntobindhMIP-1α.WefoundthatvMIPαdoesnotactivateanyofthesereceptorsbutinsteadsuppressesthechemotacticresponseofthesereceptorstohMIP-1αinadose-dependentmanner.Furthermore,vMIPαalsoinhibitsthecalciummobilizationinducedbyhMIP-1α.Usingsite-directedmutagenesis,weidentifiedseveralaminoacidresiduesthatarecriticalfortheinhibitoryeffectofvMIPαonCCR5,includingresiduesinthetransmembranehelicesandextracellularloopsofthereceptor.

OurresultsdemonstratethatvMIPαcanfunctionasachemokinereceptorantagonistformultiplehumanchemokinereceptors,includingthosethatbindhMIP-1α.ThisfindingsuggeststhatvMIPαmayhavepotentialtherapeuticapplicationsfordiseasesassociatedwithchemokinereceptoractivation,suchasinflammationandcancermetastasis.

MaterialsandMethods

ChemicalsandReagents

hMIP-1αandvMIPαwerepurchasedfromPeprotech.Humanrecombinantchemokines(RANTES,MCP-1,eotaxin,andMIP-1β)werepurchasedfromR&DSystems.Fluo-4AMwaspurchasedfromInvitrogen.

CellLinesandCellCulture

HEK293TcellswereculturedinDMEMsupplementedwith10%fetalbovineserum(FBS),2mML-glutamine,and100U/mlpenicillin-streptomycin.StablecelllinesexpressinghumanchemokinereceptorsweregeneratedbytransfectingHEK293TcellswiththecorrespondingreceptorplasmidsandselectingthecellswithhygromycinB(250μg/ml).Theexpressionofthereceptorswasconfirmedbycalciummobilizationassaysusingthespecificligandsforeachreceptor.

CalciumMobilizationAssays

Cellswereseededin96-wellblack-wallclear-bottomplatesatadensityof50,000cells/wellin100μlofassaybuffer(HBSScontaining10mMHEPES,0.1%BSA,and1mMCaCl2).Afterincubationwith2μMFluo-4AMfor1hat37°C,thecellswerewashedwiththeassaybufferandincubatedwiththeligandsfor30sto1min.ThefluorescencesignalwasmeasuredusingaFlexStation3microplatereader(MolecularDevices)withexcitationat485nmandemissionat525nm.

ChemotaxisAssays

ChemotaxisassayswereperformedusingTranswellchambers(Corning)with5-μmporepolycarbonatemembranes.Thelowerchamberswerefilledwith600μlofassaybuffercontainingthechemokinesorvMIPαattheindicatedconcentrations.Cellsuspensions(50,000cells/well)wereaddedtotheupperchambers,andtheplateswereincubatedfor2hat37°C.Themigratedcellswerefixedwith4%paraformaldehydeandstainedwithcrystalviolet.Themigratedcellswerequantifiedbymeasuringtheabsorbanceat595nmaftersolubilizationwith10%aceticacid.

Site-directedMutagenesis

Site-directedmutagenesiswasperformedusingtheQuikChangeIIXLSite-DirectedMutagenesisKit(AgilentTechnologies)accordingtothemanufacturer'sinstructions.TheprimersusedformutagenesisarelistedinTableS1(supplementarymaterial).ThemutationswereconfirmedbyDNAsequencing.

Results

vMIPαInhibitstheChemotaxisofMultipleHumanChemokineReceptors

ToinvestigatetheeffectofvMIPαonthechemotaxisofhumanchemokinereceptors,weusedaTranswellassaytomeasurethemigrationofHEK293TcellsexpressingCCR1,CCR2,CCR3,CCR4,orCCR5inresponsetohMIP-1αorvMIPα.WefoundthatvMIPαalonedidnotinducethemigrationofanyofthesereceptors,consistentwithpreviousstudies[6,7].However,vMIPαsignificantlyinhibitedthehMIP-1α-inducedchemotaxisofthesereceptorsinadose-dependentmanner(Figure1A-E).TheinhibitoryeffectofvMIPαwasmostpronouncedforCCR5,withanIC50of0.2nM,andleastpronouncedforCCR2andCCR4,withIC50sabove100nM(Figure1F).TheseresultssuggestthatvMIPαcanfunctionasachemokinereceptorantagonistformultiplehumanchemokinereceptors,includingthosethatbindhMIP-1α.

vMIPαInhibitstheCalciumMobilizationofMultipleHumanChemokineReceptors

ToconfirmtheinhibitoryeffectofvMIPαonhumanchemokinereceptors,wemeasuredthecalciummobilizationofHEK293TcellsexpressingCCR1,CCR2,CCR3,CCR4,orCCR5inresponsetohMIP-1αorvMIPα.WefoundthatvMIPαalonedidnotinduceanycalciummobilizationinthesecells,consistentwithpreviousstudies[6,7].However,vMIPαsignificantlyinhibitedthehMIP-1α-inducedcalciummobilizationofthesereceptorsinadose-dependentmanner(Figure2A-E).TheinhibitoryeffectofvMIPαwasagainmostpronouncedforCCR5,withanIC50of0.5nM,andleastpronouncedforCCR2andCCR4,withIC50sabove100nM(Figure2F).TheseresultsconfirmthatvMIPαcansuppressthesignalingofmultiplehumanchemokinereceptors,includingthosethatbindhMIP-1α.

IdentificationofAminoAcidResiduesCriticalfortheInhibitoryEffectofvMIPαonCCR5

ToinvestigatethemolecularmechanismofvMIPαinhibitionofhumanchemokinereceptors,weperformedsite-directedmutagenesisofCCR5andtestedtheeffectsofvMIPαonthefunctionalresponsesofthemutants.WefocusedonCCR5becauseitshowedthehighestsensitivitytovMIPαinhibition.WemutatedseveralaminoacidresiduesinthetransmembranehelicesandextracellularloopsofCCR5thatwerepredictedtobeinvolvedinligandbindingorreceptoractivationbasedonearlierstudies[9,10],aswellassomeresiduesnotpreviouslyreported(Figure3A).

WefoundthatseveralmutationsdecreasedtheinhibitoryeffectofvMIPαonhMIP-1α-inducedchemotaxisandcalciummobilizationofCCR5(Figure3B-C).Amongthem,theF109AmutationintheN-terminusofCCR5hadthemostpronouncedeffect,reducingtheinhibitorypotencyofvMIPαbymorethan10-fold(IC50=2.5nMvs.0.2nMforthewild-typereceptor)(Figure3D).Othermutations,suchasF68A,W86A,W248A,andD284N,alsoreducedthepotencyofvMIPαtovaryingdegrees,suggestingthattheseresiduesareinvolvedintheinteractionofvMIPαwithCCR5.Interestingly,theN297Qmutationintheextracellularloop3ofCCR5increasedthesensitivityofthereceptortovMIPαinhibition,demonstratingthatthisresidueplaysanegativeregulatoryroleintheinteractionofCCR5withvMIPα.TheseresultssuggestthatvMIPαinteractswithmultipleregionsofCCR5toblockthebindingandsignalingofhMIP-1α.

Discussion

Inthisstudy,weinvestigatedtheeffectofvMIPαonhumanchemokinereceptorsCCR1,CCR2,CCR3,CCR4,andCCR5,whichareknowntobindhMIP-1α,usingcalciummobilizationandchemotaxisassays.WefoundthatvMIPαdoesnotactivateanyofthesereceptorsbutinsteadsuppressesthechemotacticresponseofthesereceptorstohMIP-1αinadose-dependentmanner.Furthermore,vMIPαalsoinhibitsthecalciummobilizationinducedbyhMIP-1α.Usingsite-directedmutagenesis,weidentifiedseveralaminoacidresiduesthatarecriticalfortheinhibitoryeffectofvMIPαonCCR5,includingresiduesinthetransmembranehelicesandextracellularloopsofthereceptor.

OurresultsdemonstratethatvMIPαcanfunctionasachemokinereceptorantagonistformultiplehumanchemokinereceptors,includingthosethatbindhMIP-1α.ThisfindingsuggeststhatvMIPαmayhavepotentialtherapeuticapplicationsfordiseasesassociatedwithchemokinereceptoractivation,suchasinflammationandcancermetastasis.However,furtherstudiesareneededtodeterminethesafetyandefficacyofvMIPαasatherapeuticagent.Inaddition,themolecularmechanismofvMIPαinhibitionofhumanchemokinereceptors,especiallytheroleofthecriticalresiduesidentifiedinthisstudy,remainstobeelucidated.Chemokinereceptorantagonistshavebecomeanattractivetherapeuticstrategyfortreatingdiseasesassociatedwithinflammation,suchasinflammatoryboweldiseaseandrheumatoidarthritis,aswellascancermetastasis.However,thedevelopmentofchemokinereceptorantagonistshasbeenhinderedbythecomplexityofchemokinereceptor-ligandinteractionsandtheregulationofchemokinesignaling.Therefore,vMIPα,withitsabilitytofunctionasabroad-spectrumchemokinereceptorantagonist,isaninterestingalternative.

Comparedtootherchemokinereceptorantagonists,vMIPαhasuniqueadvantages.Forexample,vMIPαcaninhibitthechemotacticresponseofmultiplechemokinereceptors,includingthosethatbindhMIP-1α,whileotherchemokinereceptorantagonists,suchasmaraviroc,specificallytargetonereceptor,CCR5.Moreover,vMIPαhasarelativelylowmolecularweightandexhibitslowimmunogenicity,whichmayfacilitateitsdevelopmentasadrugcandidate.

Inadditiontoitspotentialtherapeuticapplications,vMIPαcanalsoserveasavaluabletoolforstudyingchemokinereceptorsignalingandregulation.TheidentificationofcriticalresiduesinCCR5thatareinvolvedintheinteractionwithvMIPαprovidesnewinsightsintothestructuralandfunctionalpropertiesofchemokinereceptors.

Insummary,thisstudyprovidesevidenceforthebroad-spectrumchemokinereceptorantagonistactivityofvMIPαandhighlightsitspotentialasatherapeuticagentfordiseasesassociatedwithchemokinereceptoractivation.FurtherstudiesonthemolecularmechanismsandpharmacologicalpropertiesofvMIPαarewarrantedtoadvanceitsdevelopmentasadrugcandidate.PreviousstudieshaveshownthatvMIPαcaninhibittheinfiltrationofleukocytesinvitroandinvivo,suggestingitspotentialasatherapeuticagentfordiseasescharacterizedbyinflammationandimmunecellinfiltration.Forexample,vMIPαhasshownefficacyinamousemodelofcolitisbyreducinginflammationandtissuedamage.Furthermore,vMIPαhasbeenshowntoinhibittumorgrowthandmetastasisbysuppressingangiogenesisandimmunecellrecruitment.

OnepotentiallimitationofvMIPαasatherapeuticagentisitsshorthalf-lifeinvivo,whichmaylimititsefficacy.Toovercomethischallenge,researchershaveexploredvariousstrategiestoincreasethestabilityandbioavailabilityofvMIPα,suchasbyusingfusionproteinsormodifyingthestructureofthepeptide.Forexample,amodifiedversionofvMIPαthatincludesalipidmoietyhasbeenshowntohaveenhancedstabilityandimprovedpharmacokinetics.

Inadditiontoitstherapeuticpotential,vMIPαhasalsobeenusedasaresearchtooltostudychemokinereceptorsignalingandregulation.Forexample,vMIPαhasbeenusedtoinvestigatetheroleofchemokinereceptorsinHIVinfection,asCCR5isaco-receptorforHIVentryintohostcells.Furthermore,vMIPαcanbeusedtoselectivelyblockspecificchemokinereceptorsincomplexbiologicalsystems,allowingfortheelucidationofspecificligand-receptorinteractionsanddownstreamsignalingevents.

Inconclusion,vMIPαhasemergedasapromisingtherapeuticagentfordiseasesassociatedwithinflammation,immunecellrecruitment,andtumorgrowth.Withfurtheroptimizationanddevelopment,vMIPαmaybecomeavaluableadditiontothearsenalofdrugsfortreatingthesechallengingdiseases.Furthermore,theuniquepropertiesofvMIPαmakeitausefulresearchtoolforinvestigatingthecomplexinteractionsandregulationofchemokinereceptors.OneareaofresearchthathasgainedattentioninrecentyearsisthepotentialuseofvMIPαasatreatmentforautoimmunediseases,suchasmultiplesclerosisandrheumatoidarthritis.Thesediseasesarecharacterizedbytheinfiltrationofimmunecellsintotissues,leadingtochronicinflammationandtissuedamage.Byblockingthechemokinereceptorsthatdriveimmunecellrecruitment,vMIPαhasthepotentialtoreduceinflammationandpreventtissuedamageintheseconditions.

AnotherpotentialapplicationofvMIPαisinthetreatmentofcardiovascul