ADAMTS(adisintegrinandmetalloproteinasewiththrombospondinmotif)aremultidomainextracellularproteinases,containingatleastonethrombospondintype-1repeat[1].Theenzymesareinvolvedinprocessingofextracellularmatrixproteinsandproteinsinthecirculation.ADAMTS5wasfirstpurifiedfrombovinenasalcartilageconditionedmediaandhumanADAMTS5CDNAwasclonedfromahumanlivercDNAlibrary[2].ADAMTS5consistsofaprodomain,acatalyticdomain,adisintegrindomain,athrombospondintype-1motif,aCys-richregionfollowedbyaspacersequenceandthrombospondinsubmotifs.Theprodomainismostlikelycleaved-offbyafurin-typeenzymebeforeADAMTS5isreleasedfromcells.
ADAMTS5isexpressedconstitutivelyinsynovium[3]anditdegradesaggrecan,themajorproteoglycanofarticularcartilage[4].LikeADAMTS4,anothermemberoftheADAMTS-family,ADAMTS5cleavesaggrecanat5sites[4].Fourcleavagesitesarelocatedinthechondroitinsulfate-richregionbetweenaggrecanglobulardomainsG2andG3,whileonesiteiscontainedintherod-shapedpolypeptidebetweenglobulardomainsG1andG2.Inaddition,ADAMTS5cleavesonemoresiteinthechondroitinsulfate-richregionthatisnotcleavedbyADAMTS4[4].ADAMTS5isinhibitedbyTIMP3[5,6]and2-macroglobulin[7].
Purity:AsjudgedfromSDS-PAGEtruncatedADAMTS5represents50%oftotalproteinofthepreparation.Theproteinconcentrationis0.1mg/ml.
SpecificActivity:AggrecanaseactivityoftheADAMTS5preparationisdeterminedwithrecombinantaggrecaninterglobulardomain(Aggrecan-IGDfromMDBiosciences).ADAMTS4hydrolyzesthe“aggrecanase”sitewithinthisdomain(peptidebondE373-A374inhumanaggrecan). Therecombinantsubstrateisincubatedataconcentrationof0.1μMwithADAMTS5in50mMTris-HCl,pH7.5,150mMNaCl,5mMCaCl2,1μMleupeptin,1μMpepstatin,1mMPefabloc,0.05%Brij35for15minat37°C.AseriesofADAMTS5dilutionsrangingfrom103-to105-foldistested.Substratecleavageatthe“aggrecanase”-siteisestimatedfromtheappearanceofthehydrolysisfragmentwiththenovelN-terminusARGSVIL.Thefragmentisquantifiedwithtwomonoclonalantibodies,onedirectedagainsttheneoepitopeARGSVIL,theotheragainstthesequenceC-terminalfromtheneoepitope(InviLISAAggrecanaseActivityAssay).Underthespecifiedconditionsthehydrolysisrateis>0.5nmoleshydrolyzedsubstrate/minxmlADAMTS5preparationor>5nmoleshydrolyzedsubstrate/minxmg.
Inhibitors:ADAMTS5isinhibitedbytissueinhibitorofmatrixmetalloproteinases3(TIMP3)andby2-macroglobulin(seesection3).EnzymeactivityisalsosuppressedbychelatorsofdivalentcationsasEDTAandbysyntheticmetalloproteinaseinhibitors.
StABIlity&Storage:RecombinantADAMTS5isstableuntiltheexpirydategivenonthelabelwhenstoredat-70°C.Theenzymecanbekeptat-20°Cforseveralweeks.Repeatedfreezingandthawingshouldbeavoided.
Applications:RecombinantADAMTS5isusedtostudythedegradationofextracellularmatrixproteoglycans,toscreenforinhibitorsofADAMTS5andtocharacteriseinhibitoractions.Theenzymepreparationcanalsobeutilizedinenzymaticandimmunochemicalassays.
