SecretedhumanHtrA1serineproteaseissupposedtoformoligomersofidenticalproteinchainsasrevealedbyX-rayanalysisforthebacterialHtrAproteinDegP[1].PolypeptidechainsofhumanHtrA1consistofseveraldomains:AnN-terminalinsulin-likegrowthfactordomainisfollowedbyaKazal-typeserineproteaseinhibitordomain,alinkerregion,atrypsin-likeproteasedomainandaPDZdomain.ThefunctionofHtrA1appearscloselylinkedtosignalingbyproteinsoftheTGFßfamily.Duringmouseembryodevelopment,HtrA1islocalizedinspecificareaswheresignalingbyTGFßfamilyproteinsoccurs.HtrA1bindsTGFß,BMP4,Gdf5andactivin.Itinhibitssignalingbythesefactors.ForinhibitionserineproteaseactivityofHtrA1isessential.HtrA1isupregulatedinosteoarthriticcartilage.IncreasedcartilageHtrA1maypossIBLyaggravateosteoarthritisbyantagonizingTGFßandtherebypromotingterminaldifferentiationofarticularchondrocytes.Inlaterstagesoftumorprogression,HtrA1isdownregulated.Asaprotease,HtrA1hydrolyzestypeIIprocollagenα1C-propeptide,decorinandbiglycan.ProteolyticactivityofHtrA1isregulatedbyligandbindingtothePDZdomain.

MolecularForm:

RecombinanthumanHtrA1isexpressedininsectcellswithaC-terminalHis-tagandpurified

frominsectcellculturesupernatants.ThecalculatedMrofsecretedHtrA1is50kDa.HtrA1is

solublizedin50mMTris-HCl,pH7.5,150mMNaCl,5mMCaCl2,0.05%Brij-35.

Purity:

RecombinantHtrA1appearsasamajorproteinofabout53kDinSDS-PAGE(>80%oftotal

protein).MinorbandsofHtrA1fragmentsmaybevisibleintheenzymepreparation.

EnzymaticActivity:

ProteolyticactivityofrecombinanthumanHtrA1isdocumentedbydigestionofß-casein.0.5

mgß-casein/mlarecompletelydigestedby5μg/mLHtrA1within3hoursat37ºC(seefigure

above).