Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue ...

19 Pages·2007·1.02 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.282,NO.4,pp.2405–2422,January26,2007
©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Expression and Functional Characterization of the
Cancer-related Serine Protease, Human Tissue Kallikrein 14*
Receivedforpublication,August31,2006andinrevisedform,November14,2006 Published,JBCPapersinPress,November16,2006,DOI10.1074/jbc.M608348200
CarlaA.Borgon˜o‡,IacovosP.Michael‡,JulieL.V.Shaw‡,Liu-YingLuo‡,ManikC.Ghosh‡,AntoninusSoosaipillai‡,
LindaGrass‡,DionyssiosKatsaros§,andEleftheriosP.Diamandis‡1
Fromthe‡DepartmentofPathologyandLaboratoryMedicine,MountSinaiHospitalandtheDepartmentofLaboratoryMedicine
andPathobiology,UniversityofToronto,Toronto,OntarioM5G1X5,Canadaandthe§DepartmentofGynecology,
GynecologicOncologyUnit,UniversityofTurin,Turin10126,Italy
Humantissuekallikrein14(KLK14)isanovelextracellular presence of a nucleophilic serine residue at the active site,
serineprotease.ClinicaldatalinkKLK14expressiontoseveral account for 30% of all proteases within the human
diseases,primarilycancer;however,littleisknownofits(patho)- degradome (1). Based on structural homology, SP are cate-
physiological role. To functionally characterize KLK14, we gorizedinto12clans,themosthighlypopulatedbeingclan
expressedandpurifiedrecombinantKLK14inmatureandpro- PA(S),andarefurtherclassifiedintofamiliesthatsharehigh
enzymeformsanddetermineditsexpressionpattern,specific- sequencesimilarity(2).
ity,regulation,andinvitrosubstrates.Byusingournovelimmu- Humantissuekallikreins(KLK,EC3.4.21)formasubgroup
noassay,thenormaland/ordiseasedskin,breast,prostate,and of15secreted(chymo)tryptic-likeSPwithintheS1Afamilyof
ovary contained the highest concentration of KLK14. Serum clanPA(S)andareencodedbythelargestcontiguousclusterof
KLK14 levels were significantly elevated in prostate cancer proteasegenesintheentiregenome,locatedonchromosome
patientscomparedwithhealthymales.KLK14displayedtryp- 19q13.4 (1, 3, 4). Because KLKs are widely expressed, KLK
sin-like specificity with high selectivity for P1-Arg over Lys. activityisimplicatedinanassortedarrayofnormalphysiolog-
KLK14 activity could be regulated as follows: 1) by autolytic icalprocesses(e.g.bloodpressureregulation,skinhomeostasis,
cleavageleadingtoenzymaticinactivation;2)bytheinhibitory andsemenliquefaction)andinthepathobiologyofseveraldis-
serpins (cid:1)-antitrypsin, (cid:1)-antiplasmin, antithrombin III, and eases(e.g.cancer,neurodegenerativedisorders,anddermato-
1 2
(cid:1)1-antichymotrypsinwithsecondorderrateconstants(k(cid:2)2/Ki) ses) (3–5). Because of the frequent dysregulation of KLK
of49.8,23.8,1.48,and0.224(cid:3)M(cid:4)1min(cid:4)1,respectively,aswellas expressioninmalignancy,KLKshavebeenintenselystudiedin
plasminogen activator inhibitor-1; and 3) by citrate and zinc terms of their clinical applicability as cancer biomarkers. In
ions,whichexertedstimulatoryandinhibitoryeffectsonKLK14 additiontohumankallikrein3(KLK3,alsoknownasprostate-
activity,respectively.Wealsoexpandedtheinvitrotargetrep- specificantigen(PSA)),thepremierbiomarkerinclinicalmed-
ertoireofKLK14toincludecollagensI–IV,fibronectin,laminin, icineforprostatecancer(6),mostotherKLKshaveemergedas
kininogen, fibrinogen, plasminogen, vitronectin, and insulin- promisingmarkersofdiagnosis,prognosis,predictionofther-
likegrowthfactor-bindingproteins2and3.Ourresultsindicate apeuticresponse,andmonitoringforseveralcancertypes,par-
thatKLK14maybeimplicatedinseveralfacetsoftumorpro- ticularlyovariancarcinoma(5).
gression,includinggrowth,invasion,andangiogenesis,aswell AlongwithsevenotherKLKgenes,humantissuekallikrein
asinarthriticdiseaseviadeteriorationofcartilage.Thesefind- gene14(KLK14)wasindependentlyidentifiedinourlaboratory
ingsmayhaveclinicalimplicationsforthemanagementofcan- bypositionalcandidatecloningin2001(7,8).Subsequentstud-
cerandotherdisordersinwhichKLK14activityiselevated. ies demonstrated that the KLK14 gene is under steroid-hor-
mone regulation (9, 10) and is most highly expressed in the
glandularepitheliaofthebreast(10,11)andprostate(8,10)and
Proteasesincludeagroupofenzymesthatcatalyzepeptide intheepidermis(i.e.stratumgranulosum)andappendages(i.e.
bondhydrolysis.Serineproteases(SP),2characterizedbythe hairfollicularepitheliumandeccrinesweatglands)oftheskin
(10,12,13).Consequenttoitssecretion,KLK14formsacon-
stituent of seminal plasma (10) and sweat (14) and is present
*Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment
in its catalytically active form in the stratum corneum of the
ofpagecharges.Thisarticlemustthereforebeherebymarked“advertise-
ment”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. skin (13–15). Furthermore, aberrant KLK14 expression has
1Towhomcorrespondenceshouldbeaddressed:Dept.ofPathologyand beendetectedinpatientswithbreast(7,10,11,16),ovarian(7,
LaboratoryMedicine,MountSinaiHospital,600UniversityAve.,Toronto,
9, 10), prostate (7, 17), and testicular (7) cancers and peeling
OntarioM5G1X5,Canada.Tel.:[phone];Fax:[phone];E-mail:
[email protected]. skinsyndrome(18),atthetissueand/orserumlevel.Correlative
2Theabbreviationsusedare:SP,serineproteases;ACT,(cid:1)-antichymotrypsin; clinicaldatahavealsolinkedelevatedKLK14mRNAexpression
1
AP, (cid:1)-antiplasmin; AS , angiostatin4.5; ATIII, antithrombin III; BisTris,
2 4.5
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ECM,
extracellular matrix; IGFBP, insulin-like growth factor-binding protein;
HPLC,highperformanceliquidchromatography;KLK,kallikrein;LMWK, cificantigen;ELISA,enzyme-linkedimmunosorbentassay;DMEM,Dulbec-
low molecular weight kininogen; MMP, matrix-metalloprotease; PAI-1, co’smodifiedEagle’smedium;FBS,fetalbovineserum;AMC,7-amino-4-
plasminogenactivatorinhibitor;RSL,reactivesiteloop;serpin,serinepro- methylcoumarin; ACN, acetonitrile; IGF, insulin-like growth factor; Tos,
teaseinhibitor;MES,4-morpholineethanesulfonicacid;PSA,prostate-spe- tosyl;Boc,t-butoxycarbonyl;Suc,succinyl;AT,(cid:1)-antitrypsin.
1
This is an open access article under the CC BY license.
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2405
EnzymaticActionofKLK14
withaggressiveformsofbreast(11,16)andprostatecancer(17) sites between the 5(cid:1) promoter and the 3(cid:1) terminator of the
and with the prognosis of breast (16) and ovarian (9) cancer AOX1gene,in-framewiththeyeast(cid:1)-matingfactor(forsecre-
patients. Thus, KLK14 represents a potential biomarker and tion), using standard techniques. The PmeI-linearized
therapeutictargetforseveralpathologicconditions. pPICZ(cid:1)A-KLK14 construct was transformed into chemically
AswithallS1AfamilySP,KLK14issynthesizedasaninactive competentP.pastorisyeaststrainsX-33,GS115,andKM71.A
precursororzymogen(hereindenotedpro-KLK14),containing stable X-33 transformant was selected, and recombinant
a 6-amino acid N-terminal pro-peptide that maintains its KLK14 expression was induced with 1% methanol/day for 6
latency(7,8).Proteolyticremovalofthepro-peptideatLys24– daysat30°Cinashakingincubator(250rpm).
Ile25isrequiredtogenerateactiveKLK14,aprocessthatmaybe Mature recombinant KLK14 was purified to homogeneity
performedbyKLK5(19).BecauseofthepresenceofAsp198in fromculturesupernatantbyatwo-stepprocedureconsistingof
itsS1bindingpocket(7,8),KLK14ispredictedtoexerttrypsin- cation-exchangeandaffinitychromatography.Typically,1liter
likesubstratespecificitywithapreferenceforbasicP1residues ofculturesupernatantwasclarifiedbycentrifugationandcon-
(Schechter and Berger nomenclature (20) is used to describe centrated20-foldbypositivepressureultrafiltrationinanAmi-
theinteractionbetweenproteasesubsites(Sn-S1;S1(cid:1)–Sn(cid:1))and conTMstirringchamber(MilliporeCorporation,Bedford,MA)
corresponding substrate residues (Pn-P1;P1(cid:1)–Pn(cid:1)), where witha10-kDacutoffnitrocellulosemembrane(Millipore).The
P1–P1(cid:1)denotesthescissilebond).However,we(21)andothers concentratedsupernatantwasdiluted1:4in10mMMES(pH
(19),throughtheuseofphage-displaytechnologyandchromo- 5.3) and fractionated on a pre-equilibrated 5-ml cation-ex-
genicsubstrates,respectively,haverecentlydemonstratedthat change HiTrapTM SP HP column (GE Healthcare), with the
KLK14canaccommodatebothbasic(ArgandLys)andhydro- automated A´KTA FPLC system (GE Healthcare). Adsorbed
phobic(Tyr)P1residuesattheS1subsitewithapreferencefor KLK14waselutedwithamultistepsaltgradientusing1MKCl
Arg. Hence, KLK14 manifests dual, trypsin-like and chymo- in10mMMES(pH5.3)ataflowrateof3ml/minasfollows:(a)
trypsin-like,substratespecificity. continuouslineargradientof0–0.3MKClfor17min,(b)main-
Todate,putativebiologicalsubstratesand(patho)physiolog- tenanceat0.3MKClfor20min,(c)followedbyacontinuous
ical roles for KLK14 have been inferred from its expression lineargradientfrom0.3–1MKClfor50min.Elutionfractions
pattern, substrate specificity, and the known function of co- (3ml)containingKLK14wereidentified(asdescribedbelow),
localizedKLKs.Phage-displayedpentapeptidemotifspreferred pooled,andconcentratedusingBiomax-10Ultrafree(cid:1)-15cen-
byKLK14wereidentifiedwithinseveralintactproteins,includ- trifugalfilterdevice(MilliporeCorp.,Bedford,MA).Thecon-
ingtheextracellularmatrix(ECM)moleculeslaminin,collagen centrated fractions were diluted 1:4 in 100 mM Tris-HCl (pH
typeIV,andmatrilin-4(21).Thelattermaysupportarolefor 7.8)bindingbufferandincubatedwith1mlofsoybeantrypsin
KLK14incancerprogressionviaECMdigestion,particularlyin inhibitor-agarose beads (Sigma) overnight at 4°C. The beads
breast,ovarian,andprostatetumorsthatbearelevatedKLK14 were then packed into an Econo-Pac open column (Bio-Rad)
levels. Because of its presence and prominent activity in the andwashedthreetimeswithbindingbuffer.KLK14waseluted
stratumcorneumoftheskin(13–15),KLK14isalsoimplicated with0.1Mglycinebuffer(pH3.0).
in epidermal desquamation (i.e. shedding) via degradation of Pro-KLK14—First-strandcDNAsynthesiswasperformedby
intercellular(corneo)desmosomaladhesionmoleculesthatlink reversetranscriptaseusingtheSuperscriptTMpreamplification
system(Invitrogen)with2(cid:2)goftotalhumancerebellumRNA
adjacent corneocytes (19). More recently, our group has
(Clontech)asatemplate.ThecDNAencodingprepro-KLK14
reported that KLK14 may also mediate pleiotropic effects by
(amino acids 1–251 based on GenBankTM accession number
signaling through proteinase-activated receptors 1, 2, and 4
AAK48524) was PCR-amplified in a 50-(cid:2)l reaction mixture
(22).KLK14mayexerciseitsfunctionsaloneorinaproteolytic
containing1(cid:2)lofcerebellumcDNAasatemplate,100ngof
cascade pathway, as KLK5 may activate pro-KLK14 and vice
primers(forward,5(cid:1)CACCATGTTCCTCCTGCTGACA
versa(19).
GCACTT;reverse,5(cid:1)AGACCATCATTTGTCCCGCAT
Collectively,studiesonKLK14revealthatthisnovelSPmay
CGT TTC CT, containing CACC sequence required for
playimportant(patho)physiologicalrolesatthemainsitesofits
TOPO(cid:1)cloning(underlined)andthenativeKLK14stopcodon
expression and bears clinical utility. To gain further insights
into the biological significance of KLK14 action, this study (italics)),10mMTris-HCl(pH8.3),50mMKCl,1.5mMMgCl2,
examinesKLK14expression,specificity,activityagainstcandi- 200 (cid:2)M deoxynucleoside triphosphates (dNTPs), and 0.75 (cid:2)l
(2.6 units) of Expand Long Template PCR polymerase mix
datesubstrates,andseveralmodesofregulation.
(Roche Diagnostics) on an Eppendorf master cycler (Eppen-
dorf, Westbury, NY). PCR conditions were 94°C for 2 min,
EXPERIMENTALPROCEDURES
followedby94°Cfor30s,60°Cfor30s,72°Cfor30sfor40
Cloning,Expression,andPurificationofRecombinantKLK14
cycles,andafinalextensionat68°Cfor7min.ThePCRprod-
MatureKLK14—MaturerecombinantKLK14wasproduced uct was cloned into the mammalian expression vector
intheEasyselectTMPichiapastorisexpressionsystem(Invitro- pcDNA3.1TMD/V5-His-TOPO(cid:1) (Invitrogen) using the
gen), as described previously (21). Briefly, PCR-amplified pcDNA3.1TMDirectionalTOPO(cid:1)expressionkit(Invitrogen),
KLK14 cDNA encoding the mature enzyme form of KLK14 accordingtothemanufacturer’sprotocol.TheKLK14sequence
(aminoacids25–251basedonGenBankTMaccessionnumber within the construct, denoted pcDNA3.1-KLK14, was con-
AAK48524) was cloned into P. pastoris expression vector firmed with an automated DNA sequencer using vector-spe-
pPICZ(cid:1)A (Invitrogen) at EcoRI and XbaI restriction enzyme cificprimersinbothdirections.
2406 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
EnzymaticActionofKLK14
Humanembryonickidney(HEK)-293cells(AmericanType DetectionofRecombinantKLK14
CultureCollection(ATCC),Manassas,VA)werestablytrans- To monitor recombinant KLK14 production, purification,
fectedwith3(cid:2)gofPmeI-linearizedpcDNA3.1-KLK14bylipo- and/oractivity,sampleswereanalyzedbySDS-PAGE,Western
fectionusingPolyFecttransfectionreagent(Qiagen,Valencia, blot,andzymography.
CA),accordingtothemanufacturer’sinstructions.Transfected SDS-PAGE—SDS-PAGEwasperformedusingtheNuPAGE
HEK-293cellswereincubatedinDulbecco’smodifiedEagle’s BisTris electrophoresis system and precast 4–12% gradient
medium (DMEM; Invitrogen) supplemented with 10% fetal polyacrylamidegelsat200Vfor30min(Invitrogen).Proteins
bovineserum(FBS),inahumidifiedatmospherecontaining5% werevisualizedwithaCoomassieG-250stainingsolution,Sim-
CO .After48h,cellswerere-platedinto35-mmdishes,andthe plyBlueTMSafeStain(Invitrogen),and/orbysilverstainingwith
2
selection agent Geneticin (G418, 300 (cid:2)g/ml final concentra- theSilverXpressTMkit(Invitrogen),accordingtothemanufac-
tion;Invitrogen)wasadded.Resistantcloneswerechosenover turer’sinstructions.
14 days under continuous G418 selection and cultured sepa- Western Blots—For immunodetection of KLK14, proteins
ratelyin24wellsfollowedby6-wellplates.Oneclone,denoted resolved by SDS-PAGE were subsequently transferred onto a
P1–4,wasselectedforlargescaleexpressionofpro-KLK14and Hybond-CExtranitrocellulosemembrane(GEHealthcare)at
growninDMEMwith10%FBSuntil80%confluency.Atthis 30 V for 1 h. The membrane was blocked with Tris-buffered
stage, the media were replaced with serum-free CD Chinese saline/Tween(0.1mol/literTris-HClbuffer(pH7.5)contain-
hamsterovarymediasupplementedwithGlutaMaxTMI(8mM ing0.15mol/literNaCland0.1%Tween20)supplementedwith
final concentration; Invitrogen). Conditioned media was col- 5%nonfatdrymilkovernightat4°CandprobedwithaKLK14
lectedbycentrifugationafter10daysofincubation. polyclonalrabbitantibody(producedin-house;diluted1:2000
Recombinantpro-KLK14waspurifiedtohomogeneityfrom in Tris-buffered saline/Tween) for 1 h atroom temperature.
the conditioned serum-free media of HEK-293 cells stably Themembranewaswashedthreetimesfor15minwithTris-
transfectedwithpcDNA3.1-KLK14sequentiallybycation-ex- bufferedsaline/Tweenandtreatedwithalkalinephosphatase-
changeandreversed-phasechromatography.Priortopurifica- conjugatedgoatanti-rabbitantibody(1:10,000inTris-buffered
tion,1literofmediawaspreparedasdescribedaboveforyeast saline/Tween;JacksonImmunoResearch)for1hatroomtem-
culture supernatant. Concentrated CM was diluted 1:4 in 10 perature.Finally,themembraneswerewashedagainasabove,
mMMES(pH5.3)andappliedtoapreviouslyequilibrated5-ml andfluorescencewasdetectedonx-rayfilmusingchemilumi-
SP HP-Sepharose column (GE Healthcare), previously equili- nescentsubstrate(DiagnosticProductsCorp.,LosAngeles).
bratedwith10mMMES(pH5.3),ontheA´KTAfastperform- Zymography—The proteolytic activity of recombinant
anceliquidchromatographysystemataflowrateof1ml/min. KLK14proteinswasvisualizedbygelatinzymography(Invitro-
Pro-KLK14waselutedwithamultistepsaltgradientof1MKCl gen).RecombinantKLK14wasdiluted1:1inTris-glycineSDS
in10mMMES(pH5.3)ataflowrateof3ml/minasfollows:(a) samplebufferandelectrophoresedonprecastNovex(cid:1)10%gel-
continuous linear gradient of 0–0.25 M KCl for 17 min, (b) atinzymogramgels(Invitrogen)at125Vfor2.5hat4°C.The
maintenance at 0.25 M KCl for 33 min, (c) linear gradient of gels were subsequently incubated in zymogram renaturing
0.25–0.35MKClfor8.3min,(d)maintenanceat0.35MKClfor buffer(Invitrogen)fortwo30-minintervalsatroomtempera-
33min,and(e)followedbyalineargradientof0.35-0.8MKCl ture, followed by incubation in zymogram developing buffer
for33min.Elutionfractions(3ml)wereanalyzedbyournewly (Invitrogen) for 3 h at 37°C.Gels were stained with Simply-
developed enzyme-linked immunosorbent assay (ELISA; BlueTMSafeStainanddestaineduntilthewhitelyticbandscor-
describedherein)andbydetectionmethodsmentionedbelow. respondingtoareasofproteaseactivitywerevisible.
Fractions containing pro-KLK14 were then pooled, concen-
trated,andsupplementedwithafinalconcentrationof1%trif- GenerationofAntibodiesagainstKLK14
luoroaceticacidandloadedin0.1%trifluoroaceticacidinH O NewZealandWhitefemalerabbitsandBALB/cfemalemice
2
ontoa1-mlVydacC4reversed-phasecolumn(TheSeparations wererepeatedlyimmunizedwithpurifiedrecombinantmature
Group,Inc.,Hesperia,CA)connectedtoanAgilentseries1100 KLK14(100(cid:2)g)forpolyclonalandmonoclonalantibodydevel-
HPLC system equipped with a diode array detector (Agilent opment, respectively. KLK14 was diluted 1:1 in complete
Technologies,Inc,PaloAlto,CA).Elutionwasperformedwith Freund’s adjuvant for the first injection and in incomplete
a multistep acetonitrile (ACN) gradient at a flow rate of 0.8 Freund’s adjuvant for subsequent injections. Injections were
ml/minconsistingof5-minlineargradientsof20–25,25–30, repeatedthreetimesformiceandsixtimesforrabbitsat3-week
30–32, 32–35, 35–40, and 40–100% ACN intervened by intervals.Thepolyclonalseraweretestedevery2weeksbyan
15-minstepsat25,30,32,and35%ACN.ACNwasremoved immunofluorometric method described in detail elsewhere
fromeachfractionbyevaporationwithnitrogengasatapres- (10), until the highest antibody titers against KLK14 were
sureof15p.s.i. detected.
ForbothrecombinantKLK14proteins,puritywasassessed Monoclonal antibodies against KLK14 were produced by
on silver-stained SDS-polyacrylamide gels (described below). standardhybridomatechnology.Thesplenocytesfromprese-
Concentration was determined by the BCA method (Pierce), lected immunized mice were fused with the Sp2/0 myeloma
andproteinidentitywasconfirmedbytandemmassspectrom- cellsusingpolyethyleneglycol1500.Theresultanthybridoma
etry,asdescribedpreviously(23).Proteinswerealiquotedand cells were cultured in 96-well plates in DMEM (Invitrogen)
storedat(cid:2)80°Cin0.1Msodiumacetatebuffer(pH5.0). containing 20% FBS, 200 mM glutamine, 1% OPI (oxaloacetic
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2407
EnzymaticActionofKLK14
acid,pyruvicacid,insulin),and2%HAT(hypoxanthine,ami- TissueExtracts,BiologicalFluids,andCancerCellLines
nopterin,thymidine;Sigma)forselectionat37°C,5%CO for
2 TheconcentrationofKLK14innormalandcanceroustissue
10–14 days. Hybridoma supernatants were collected and
extracts, biological fluids, and conditioned media of various
screenedbyanimmunofluorometrictechniquedescribedpre-
cancer cell lines was determined with the newly developed
viously(24).Positivecloneswerethenexpandedsequentiallyin
KLK14-specificELISA.
24-and6-wellplatesincompletemedia(reducingtheFBSto
Tissue Extracts—A panel of 40 normal tissue extracts were
15% and changing the HAT to HT). Antibody isotyping was
preparedasdescribedelsewhere(10),includingadrenalgland,
subsequentlyperformedusingamouseimmunoglobulinsub-
axillarylymphnode,bone,breast,colon,endometrium,esoph-
typeidentificationkit(Sigma)toisolateclonesproducinganti-
agus,fallopiantube,kidney,liver,lung,mesentericlymphnode,
KLK14 monoclonal antibodies of the IgG class. One positive
skeletalmuscle,ovary,pancreas,prostate,seminalvesicle,skin,
hybridoma clone, denoted 2E9, was expanded in flasks in
smallintestine,spleen,stomach,testis,thyroid,trachea,ureter,
serum-freeCDChinesehamsterovarymedia(Invitrogen)con-
uterus,salivarygland,pharyngealtonsil,placenta,cervix,cere-
taining200mMglutamine,forlargescaleantibodyproduction.
bellum,frontallobecortex,hippocampus,medulla,midbrain,
Anti-KLK14 monoclonal antibody 2E9 was purified from the
occipitalcortex,pituitary,pons,temporallobecortex,andspi-
culturesupernatantusingtheAffi-GelProtein-AMAPSIIkit
nal cord. Cancerous ovarian tissues, containing (cid:3)80% tumor
(Bio-Rad),accordingtomanufacturer’sinstructions,andused
cells,wereobtainedfrompatientswithprimaryepithelialovar-
insubsequentimmunoassaydevelopment.
iancancerwhounderwentsurgeryandtreatmentforovarian
cancer at the Department of Gynecology of the University of
DevelopmentofanELISAforKLK14
Turin, Turin, Italy, between July of 1991 and April of 1999.
A“sandwich-type”ELISA,withamousemonoclonal/rabbit
Ovarian tumor cytosolic extracts were prepared as described
polyclonal configuration, was developed as follows. The
previously(26).Totalproteincontentoftheextractswasdeter-
KLK14-specificmonoclonalantibody(clone2E9;developedin-
minedusingthebicinchoninicacidmethod,withbovineserum
house) was first immobilized on a 96-well white polystyrene
albuminasastandard(Pierce).
plate(500ng/well)byincubatingincoatingbuffer(50mmol/
BiologicalFluids—Cerebrospinalfluid,breastmilk,amniotic
literTris,0.05%sodiumazide(pH7.8))overnightatroomtem-
fluid, seminal plasma, ascites fluid from ovarian cancer
perature.Theplatewasthenwashedthreetimeswithwashing
patients,breastcancercytosolicextracts,andserafrommales
buffer(50mmol/literTris,150mmol/literNaCl,0.05%Tween
and females without known disease and of prostate cancer
20(pH7.8)).KLK14standardsorsampleswerethenpipetted
patientswereresidualsamplessubmittedpreviouslyforroutine
intoeachwell(100(cid:2)l/well,diluted1:1inassaybuffer(50mmol/
biochemicaltestingandstoredat(cid:2)80°C.Investigationswere
literTris,6%bovineserumalbumin,10%goatIgG,2%mouse
carriedoutinaccordancewiththeethicalstandardsoftheHel-
IgG,1%bovineIgG,0.5mol/literKCl,0.05%sodiumazide(pH
sinki Declaration of 1975, as revised in 1983, and have been
7.8)),incubatedfor2hwithshaking,andthenwashedsixtimes,
asabove.Subsequently,100(cid:2)lofrabbitanti-KLK14polyclonal approved by the Institutional Review Board of Mount Sinai
Hospital,Toronto,Canada,andtheInstituteofObstetricsand
sera(developedin-house)diluted1000-foldinassaybufferwas
Gynecology,Turin,Italy.
added and incubated for 1 h. After incubation, the plate was
Cell Lines—The astrocytoma cell line, SW-1088 (HTB12);
washed as above, and alkaline phosphatase-conjugated goat
breastcancercelllinesMDA-MB-468(HTB132),BT-474(HTB-
anti-rabbitIgG(JacksonImmunoResearch),diluted3000-fold
20),T-47D(HTB-133),MCF7(HTB-22),andZR-75,MFM223,
in assay buffer, was applied and plates were incubated for 45
MDA-MB-231(HTB-26),MDA-MB-453(HTB-131),MDA-MB-
min.Afterwashingasabove,thealkalinephosphatasesubstrate
diflunisal phosphate (100 (cid:2)l of a 1 mM solution) in substrate 361(HTB27),BT-20(HTB19);thecolorectaladenocarcinomacell
line, COLO 320HSR (CCL-220.1); the ovarian cancer cell lines
buffer (0.1 mol/liter Tris (pH 9.1), 0.1 mol/liter NaCl, and 1
Caov-3(HTB-75),MDAH2774(CRL-10303),TOV-21G(CRL-
mmol/literMgCl )wasaddedtoeachwellandincubatedfor10
2
minfollowedbyadditionofdevelopingsolution(100(cid:2)l,con- 11730), TOV112D (CRL-11731), OVCAR-3 (HTB-161), OV-90
taining1mol/literTrisbase,0.4mol/literNaOH,2mmol/liter (CRL-11732),ES2(CRL-1978);thecervicalcancercelllinesHeLa
TbCl ,and3mmol/literEDTA)for1min.Theresultantfluo- (CCL-2),C-4I(CRL-1594),ME-180(HTB-33),HT-3(HTB-32);
3
rescence was measured by time-resolved fluorometry on the neuroblastoma cell lines SK-N-DZ (CRL-2149) and IMR-32
Cyberfluor 615 Immunoanalyzer (MDS Nordion, Kanata, (CCL-127) and neuroepithelioma cell line Sk-N-MC (HTB-10);
Ontario, Canada). Calibration and data reduction were per- thepancreaticcancercellline,MIAPaCa-2(CRL-1420);andthe
formedautomatically,asdescribedelsewhere(25). prostate cancer cell line LNCaP (CRL-1740) were purchased
The optimal assay configuration and conditions described fromATCC.TheBG-1ovariancancercelllinewaskindlypro-
above were obtained by testing several monoclonal antibody videdbyDr.HenriRochefort(MontpellierUniversity,Mont-
andpolyclonalseradilutionsanddiluents,aswellasdifferent pellier,France),andthePC-3cellline,stablytransfectedwith
incubationtimesforeachstepoftheprocedure.Assaysensitiv- androgenreceptor(PC-3(AR) );wasprovidedbyDr.Theodore
6
ity (detection limit), specificity (including cross-reactivity to Brown (Samuel Lunenfeld Research Institute, Mount Sinai
otherKLKfamilymembers),linearity,recovery,andwithin-run Hospital,Toronto,Canada).Cellswereculturedtonearconflu-
and between-run imprecisions were determined as described encyinRPMImedium(Invitrogen)supplementedwithgluta-
previously(10). mine(200nM)andFBS(100ml/liter)inplasticflasks.
2408 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
EnzymaticActionofKLK14
ProteaseAssaysandKineticConstantDeterminations played(cid:3)95%purityonCoomassieBlue-stainedpolyacrylam-
ide gels, were diluted in water to a final concentration of 1
7-Amino-4-methylcoumarin (AMC) was purchased from
mg/ml, and were stored at (cid:2)20°C. To calculate second-rate
Sigma. The following synthetic AMC substrates were obtained
constants, the inhibition of KLK14 by serpins was performed
fromBachemBioscience(KingofPrussia,PA):Boc-Phe-Ser-Arg-
under pseudo-first order conditions, as described previously
AMC(FSR-AMC),Boc-Val-Pro-Arg-AMC(VPR-AMC),H-Pro-
(28–30).KLK14waspreincubatedwitheachserpinforvarious
Phe-Arg-AMC (PFR-AMC), benzyloxycarbonyl-Gly-Gly-Arg-
lengthsoftime(0–5minforAT,AP,andPAI-1;0–10minfor
AMC (GGR-AMC), Boc-Leu-Gly-Arg-AMC (LGR-AMC),
ATIII,and0–60minforACT)andatdifferentKLK14:serpin
Boc-Leu-Lys-Arg-AMC (LKR-AMC), Boc-Leu-Arg-Arg-AMC
molar ratios at room temperature (for AT, AP, PAI-1 and
(LRR-AMC), Boc-Gln-Arg-Arg-AMC (QRR-AMC), Boc-Gln-
ATIII)or37°C(forACT)withgentleagitation.Thereaction
Ala-Arg-AMC (QAR-AMC), Boc-Gln-Gly-Arg-AMC (QGR-
was quenched by diluting 10 (cid:2)l in 190 (cid:2)l of optimal buffer
AMC), Tos-Gly-Pro-Arg-AMC (GPR-AMC), Tos-Gly-Pro-Lys-
AMC (GPK-AMC), Boc-Glu-Lys-Lys-AMC (EKK-AMC), containing0.2mMQAR-AMC.AMCfluorescencewasmeas-
uredontheWallac1420Victor2TMfluorometer,asdescribed
Boc-Val-Leu-Lys-AMC(VLK-AMC),andSuc-Ala-Ala-Pro-Phe-
above.Allexperimentswereperformedintriplicate.
AMC(AAPF-AMC).Suc-Leu-Leu-Val-Tyr-AMC(LLVY-AMC)
To monitor KLK14-serpin complex formation, KLK14 (0.5
waspurchasedfromthePeptideInstituteInc.(Osaka,Japan).All
substratesweredilutedindimethylsulfoxide(Me SO)toafinal (cid:2)g;1(cid:2)Mfinalconcentration)wasincubatedwitheachserpinat
concentrationof80mMandstoredat(cid:2)20°C. 2 molarratiosof1:1,1:2,1:5,and1:10atroomtemperaturefor30
min(forAT,AP,PAI-1andATIII)orat37°Cfor1h(forACT).
Individual AMC-peptide substrates (final concentrations
ReactionmixtureswereresolvedbySDS-PAGEunderreducing
rangingfrom0.004to2mM)wereincubatedwithrecombinant
conditions;gelswerestainedwithSimplyBlueTMSafeStain.
matureKLK14(finalconcentrationof12nM)atafinalvolume
of100(cid:2)lunderoptimalbufferconditionswithin96-wellwhite
EffectofIonsonKLK14Activity
polystyrenemicrotiterplates.Reactionmixturescontainedless
KLK14wasincubatedwitheachcationandcitrate(solutions
than5%(v/v)Me SO.InitialratesofKLK14-mediatedpeptide
2 prepared from salts of ZnCl , ZnSO , Zn(CH COO) , CaCl ,
hydrolysiswasmonitoredbymeasuringfreeAMCfluorescence 2 4 3 2 2
MgCl ,NaCl,KCl,sodiumcitrate,atfinalconcentrationsof0,
ontheWallac1420Victor2TMfluorometer(PerkinElmerLife 2
12,60,120,1200,and12,000nM)inoptimalassaybufferata
Sciences)withexcitationandemissionfilterssetat380and480
finalvolumeof100(cid:2)lfor10minat37°Cwithgentleagitation.
nm,respectively,at1-minintervalsfor20minat37°C.KLK14-
Atthispoint,QAR-AMC(atafinalconcentrationof0.5mM)
free reactions, for each peptide concentration, were used as
wasappliedtoeachreactionmixture,andAMCfluorescence
negativecontrols,andbackgroundcountsobtainedweresub-
was measured on the Wallac 1420 Victor2TM fluorometer, as
tracted from each value. A standard curve was constructed
described above. Residual KLK14 activity against QAR-AMC
usingknownconcentrationsofAMCinordertocalculatethe
afterincubationwithindividualcations/citratewascalculated.
rateoffreeAMCrelease.TheslopeoftheresultantAMCstand-
ardcurvewas19.184AMCfluorescencecounts/nMfreeAMC. ReversalofZn2(cid:2)InhibitionofKLK14byEDTA
The steady-state (Michaelis-Menten) kinetic constants (k /
cat KLK14(12nMfinalconcentration)wasincubatedaloneorwith
K )werethencalculatedbynonlinearregressionanalysisusing
m the following: (a) Zn2(cid:4) (1.2 (cid:2)M final concentration ZnCl ), (b)
Enzyme Kinetics Module 1.1 (Sigma Plot). All experiments EDTA(1mMfinalconcentration),(c)Zn2(cid:4)andEDTA,an2d(d)
wereperformedintriplicateandrepeatedatleasttwice. Zn2(cid:4)followedbytheadditionofEDTAafter20min,atafinal
volumeof100(cid:2)linoptimalassaybufferinmicrotiterplatewells
Pro-KLK14ActivationStudies
for10minat37°Cwithgentleagitation.Afterincubation,QAR-
The possible activation of pro-KLK14 by recombinant AMC (final concentration of 0.5 mM) was applied to each well.
mature KLK5 (produced in-house in P. pastoris as described AMCfluorescencewasmeasuredontheWallac1420Victor2TM
previously(27))wasmonitoredviacomplexformationbetween fluorometer,asdescribedabove.KLK14-freereactionswereused
KLK14andtheserineproteaseinhibitor(serpin)(cid:1)-antitrypsin asnegativecontrols,andbackgroundfluorescencewassubtracted
1
(AT),aninhibitorofKLK14,butnotofKLK5(27).Pro-KLK14 fromeachvalue.Allexperimentswereperformedintriplicate.
waspreincubatedwithKLK5inafinalvolumeof10(cid:2)latdif-
ferentmolarratiosandincubationtimes.ATwasthenaddedat InVitroProteolysisExperiments
a2-foldmolarexcesstoafinalvolumeof15(cid:2)l.Thereaction ThefollowingsubstrateswereincubatedwithKLK14:mouse
wasincubatedfor30minatroomtemperature.Ascontrols,AT sarcomacollagentypeI,chickensternalcartilagecollagentype
wasincubatedaloneandwithrecombinantmatureKLK14and II,calfskincollagentypeIII,humanplacentacollagentypeIV,
pro-KLK14 and with mature KLK5. Reaction mixtures were humanforeskinfibroblastfibronectin,humanplasmavitronec-
resolvedbySDS-PAGEunderreducingconditions,andresult- tin, laminin, and low molecular weight kininogen were pur-
antgelsweresilver-stained. chased from Sigma. Plasminogen from human plasma and
Inhibition Assays, Serpins—Serine protease inhibitors (ser- recombinant human insulin-like growth factor-binding pro-
pins), AT (SERPINA1), (cid:1)-antichymotrypsin (ACT; SER- teins2and3(IGFBP-2andIGPBP-3)wereobtainedfromR&D
1
PINA3),antithrombinIII(ATIII;SERPINC1),(cid:1)-antiplasmin SystemsInc.(Minneapolis,MN).
2
(AP;SERPINF2),andplasminogenactivatorinhibitor-1(PAI-1; CollagenI,II,andIII(2(cid:2)g);collagenIV,laminin,fibronectin,
SERPINE1)werepurchasedfromCalbiochem.Inhibitorsdis- kininogen(5(cid:2)g);fibrinogen,plasminogen(10(cid:2)g);andvitronec-
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2409
EnzymaticActionofKLK14
tin,IGFBP-2,andIGFBP-3(300ng)wereincubatedwithspecific
amountsofKLK14(6ngto1(cid:2)g)inoptimalassaybufferovertime
atroomtemperatureor37°Cwithgentleagitation.KLK14-free
reactionswereincludedasnegativecontrols.Reactionswereter-
minatedbyfreezinginliquidnitrogenandresolvedbyreducing
SDS-PAGE,asdescribedabove.GelswerestainedwithSimply-
BlueTMSafeStainorwiththeSilverXpressTMkit(Invitrogen).
Furthermore, KLK14 (0.4 and 0.8 (cid:2)g) was incubated with
fluorescentconjugatesofcollagentypesIandIV(2.5(cid:2)g)and
fibrinogen(3.75(cid:2)g)(MolecularProbes,Eugene,OR)inoptimal
assaybuffer(finalvolume200(cid:2)l)atroomtemperature.Fluo-
rescencewasmeasuredevery10minfor2hontheWallac1420
FIGURE 1. Purified recombinant mature and pro-KLK14. A–C, lane 1,
Victor2TMfluorometersetat492nmforexcitationand535nm mature KLK14; lane 2, pro-KLK14. A, silver-stained reducing SDS-PAGE of
foremission.Enzyme-freereactionswereusedasnegativecon- KLK14(300ng/lane).B,immunodetectionofKLK14usingaprimaryrabbit
polyclonal antibody (1:2000) generated against mature KLK14. C, gelatin
trols,andbackgroundfluorescencewassubtractedfromeach
zymographyofKLK14.IntactmatureKLK14isrepresentedbyasinglebandof
value.Allexperimentswereperformedintriplicate. 25kDa(‚);lowermolecularweightbandslikelycorrespondtoautodegrada-
tionproductsandareindicatedwitharrows(4).Pro-KLK14isvisualizedasa
N-terminalSequencing singlebandof27kDa(Œ).Zymographicanalysisrevealsthatonlymature
KLK14isactive,asexpected.M,molecularmassstandardsinkDa.
N-terminal sequence analysis was performed on KLK14-
generated fragments derived from recombinant mature
KLK14, fibronectin, collagen IV, plasminogen, kininogen,
fibrinogen,andvitronectin.KLK14wasincubatedalone(3(cid:2)g)
orwitheachintactprotein(2.5–5(cid:2)g)fordifferenttimepoints
asspecifiedbytheproteolysisexperiments,separatedbyreduc-
ingSDS-PAGE,andtransferredtoapolyvinylidenedifluoride
membrane (GE Healthcare), previously immersed in 100%
methanolat30Vfor1h.Afterthetransfer,themembranewas
removedandrinsedwithde-ionizedwater,stainedwithCoo-
massieBlueR-250(0.1%solutionin40%methanol)for5min,
andde-stainedfor5minin50%methanol.Themembranewas
then thoroughly washed with de-ionized water and air-dried.
FragmentswereexcisedandsubjectedtoautomatedN-termi-
nalEdmandegradation(31)consistingoffivecyclesofEdman
chemistry on a 492 Procise cLC sequencer (Applied Biosys-
tems,FosterCity,CA),followedbyanalysisofresultantphenyl-
thiohydantoin-derivativeresiduesonanHPLCcolumn.
RESULTS
RecombinantMatureandPro-KLK14
Recombinantmatureandpro-KLK14proteinswereproduced
intheirnativeforms,withoutfusiontags,inyeast(P.pastoris)and
mammalian(HEK-293)expressionsystems,respectively.Secreted
FIGURE2.ConcentrationofKLK14inadulthumantissueextracts.The
expressionwasachievedbycloningKLK14cDNAin-framewith amountofKLK14intissueextractsiscorrectedforthetotalproteincontent
theyeast(cid:1)-factorformatureKLK14andwiththenativeKLK14 andisexpressedasnanogramsofKLK14pergoftotalprotein.
signalsequenceinthecaseofpro-KLK14.AsmonitoredbySDS-
PAGE,Westernblotanalysisand/orELISA,matureKLK14was pro-peptide (Gln19–Lys24) and a 227-amino acid trypsin-like
detectedintheyeastculturesupernatantafter1dayofmethanol serineproteasedomain(Ile25–Lys251)withapredictedzymo-
induction,withthehighestlevelsproducedafter6days;maximal genactivationsiteatLys24–Ile25(7,8).Theresultantmassesof
pro-KLK14levelswereattainedaftera10-dayincubationperiodin bothrecombinantproteinswereconsistentwiththoseinferred
serum-free media (data not shown). After their respective two- fromtheprimaryKLK14sequence,indicatingalackofglyco-
step chromatographic purification procedures, recombinant sylation or other post-translational modifications. Purified
matureandpro-KLK14wereobtainedwith(cid:3)95%purity,asveri- matureKLK14appearedasanenzymaticallyactiveintactband
fiedbysilver-stainedpolyacrylamidegels(Fig.1A).Theyieldof of(cid:5)25kDaalongwith8lessabundantlowermolecularweight
purifiedmatureandpro-KLK14from1-literculturesupernatants bands,identifiedasproductsofautodegradation(Fig.1,lane1,
wasintherangeof1.5–3mgand25–50(cid:2)g,respectively,asdeter- andFig.4).Purifiedpro-KLK14wasvisualizedasasingle,enzy-
minedbyELISAandtotalproteinassays. maticallyinactivebandof(cid:5)27kDa,asexpected(Fig.1,lane2).
Prepro-KLK14isa251-aminoacidpolypeptideconsistingof TheidentityofbothrecombinantKLK14proteinswasveri-
a signal sequence (Met1–Ser18), followed by a 6-amino acid fied by Western blotting (Fig. 1B) and confirmed by tandem
2410 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
EnzymaticActionofKLK14
TABLE1
SummaryofKLK14levelsinvariousbiologicalfluids
KLK14concentration((cid:3)g/liter)
Biologicalfluid No.ofsamplestested Positivity
Range Mean(S.D.) Median
%
Seminalplasma 24 3.6–82.5 15.8(16.0) 13.0 100
Amnioticfluid 24 1.7–22.9 8.1(6.0) 6.0 100
Saliva 6 0.6–6.1 4.4(2.1) 5.3 100
Follicularfluid 19 0–8.8 1.27(2.13) 0.44 95
Breastmilk 18 0–18.6 1.2(0.42) 0.13 83
Serum
Female 32 0.03–0.25 0.14(0.05) 0.14 100
Male 17 0.04–0.24 0.12(0.05) 0.10 100
Cerebrospinalfluid 6 0 0 0 0
massspectrometry.InthecaseofrecombinantKLK14,partially
sequencedtrypticpeptidesIITGGHTCT,QVTHPNYNSR,and
QACASPGTSCR corresponded to amino acid sequences
25–33,97–106and134–144,respectively,oftheKLK14pro-
teinsequence.Likewise,sequencedpeptidefragmentsobtained
from the pro-KLK14 sample included IIGGHTCTR, SSQP-
WQAALLAGPR, QVTHPNYNSR, AVRPIEVTQACASPGT-
SCR,andDSCQGDSGGPLVCR,whichmatchedpreciselywith
KLK14aminoacidsequences25–33,34–47,97–106,126–144,
198–211, respectively (numbering is based on GenBankTM
accessionnumberAAK48524).
DistributionofKLK14inTissues,Fluids,andCellLines
A novel KLK14-specific ELISA was established to quantify
KLK14levelsinnormalandcanceroustissueextracts,biological
fluids,andcelllines.TheoptimizedKLK14-ELISA,consistingof
monoclonalantibody2E9(captureantibody)andpolyclonalrab-
bitantiserum(detectionantibody)generatedagainstrecombinant
matureKLK14,issensitive(detectionlimitof0.05(cid:2)g/liter),spe- bFIuGtUioRnEo3f.EKlLeKv1a4tiloenveolsfK((cid:2)LgK/1li4teinr)sinertuhmesoefrparoofsthaetaeltchaynmcearlepsat(nieonrmts.aDl)iastnrdi-
cificforKLK14((cid:6)0.1%cross-reactivitywithotherKLKs),recog- patientswithprostatecancer.ThepvaluewasdeterminedbytheMann-
WhitneyUtest.Horizontallinesrepresentthemedianvalues.
nizesbothmatureandpro-KLK14recombinantproteins,andis
linearfrom0.05to10(cid:2)g/literwithbetween-runandwithin-run
coefficientsofvariationof(cid:6)10%. Amongthecelllinesstudied,KLK14wasonlydetectedinthe
Amongthe40normaladulthumantissuesscreenedforKLK14 conditionedmediaofanontumorigenickeratinocytecellline
content, 18 contained detectable levels of KLK14 (Fig. 2). The (HaCaT;0.275(cid:2)g/liter)andasubsetofneuroblastoma(Sk-N-
highestKLK14levelswerefoundinskin(245ng/gtotalprotein), MC,IMR32;0.949and0.193(cid:2)g/liter,respectively),breastcan-
followedbybreast(224ng/gtotalprotein)andprostatic(131ng/g cer (MCF7, MDA-MB-468; 0.116 and 0.497 (cid:2)g/liter, respec-
total protein) tissue extracts, in general accord with previous tively), cervical cancer (C-4 I, ME-180, HT-3; 0.158–0.456
studies (7;8;10;11;13–15;17). Comparatively lower levels were (cid:2)g/liter),andovariancancer(TOV-21G,MDAH2774,OV-90,
observed in axillary lymph nodes (62.8 ng/g total protein) and TOV112D;0.103–0.604(cid:2)g/liter)celllines.
medulla (59.2 ng/g total protein) extracts with minor amounts
((cid:6)50ng/gtotalprotein)intheremainingtissues. AutodegradationofKLK14inVitro
TheconcentrationofKLK14inbiologicalfluidsfromhealthy PurifiedrecombinantmatureKLK14consistedofamixture
individuals was quantified (Table 1). In agreement with our ofpeptidesvisualizedasmultiplebandsunderreducingSDS-
priorstudy(10),thehighestlevelsofKLK14weremeasuredin PAGE (Fig. 4A). Western blot (Fig. 1B) and N-terminal
seminal plasma (15.8 (cid:7) 16.0 (cid:2)g/liter), followed by amniotic sequenceanalysisconfirmedtheidentityofthelowmolecular
fluid(8.1(cid:7)6.0(cid:2)g/liter)andsaliva(4.4(cid:7)2.1(cid:2)g/liter).Follicu- weight ((cid:6)25 kDa) protein bands as internal fragments of
larfluidandbreastmilkcontainedlowerconcentrations((cid:5)1.2 mature KLK14 likely arising from auto-proteolytic cleavage
(cid:2)g/liter). KLK14 was not detected in the cerebrospinal fluid (Fig.4).AllKLK14cleavagesitesoccurredafterP1-Argresidues
samplesexamined.Comparedwithnormal,KLK14levelswere (Fig.4B),andthemajorityresideinexposed,solvent-accessible
elevatedintheserumofpatientswithprostatecancer(Fig.3),a surfaceloops,whicharemostsusceptibletoproteolyticcleav-
likelyconsequenceofKLK14overexpressionandsecretionby age(Fig.4C).AmongKLK14autodegradationfragments,only
prostatetumortissues(17).WealsodetectedKLK14inascites fragment I retains all residues of the catalytic triad (His67,
fluidsfromovariancancerpatients(0.34(cid:7)0.7(cid:2)g/liter)aswell Asp111,andSer204;seeFig.4B)andmayexhibitresidualserine
as in cytosolic extracts of ovarian (1.77 (cid:7) 5.13 (cid:2)g/liter) and protease activity. However, prolonged incubation (24 h) of
breast(0.26(cid:7)1.17(cid:2)g/liter)tumors. matureKLK14at37°Cresultedincompletehydrolysisofthe
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2411
EnzymaticActionofKLK14
FIGURE4.AutodegradationofKLK14.A,CoomassieBlue-stainedSDS-polyacrylamidegeloffragmentedmaturerecombinantKLK14(5(cid:2)g).Intact
KLK14correspondstothe25-kDaband.LowermolecularweightautodegradationfragmentsofKLK14arelabeledI–VII.Theregionthateachpeptide
fragmentspanswithintheKLK14proteinsequenceisinparentheses.NotethatproteinbandsIIIandVIIarecomprisedof2and3individualpeptides,
respectively.M,molecularmassstandardsinkDa.B,locationoftheN-terminalsequenceofeachKLK14degradationfragmentwithintheprimaryKLK14
proteinsequence.Prepro-KLK14isformedofasignalpeptide(italics),followedbyapro-peptide(underlined)andthematuresequencecontainingthe
serineproteasedomain(boldface).Catalytictriad(His67,Asp111,andSer204)residuesareindicatedwithanasterisk.TheN-terminalsequenceofKLK14
degradationfragmentsisboxed,withthecorrespondinglabelabove.Cleavagesitesareindicatedbyanarrow;theP1aminoacidisnumbered.AandB,
KLK14sequencesarenumberedfromtheNterminusofprepro-KLK14basedonGenBankTMaccessionnumberAAK48524.C,locationofautolytic
cleavagesiteswithinthetheoreticaltertiarystructureofmatureKLK14,aspredictedbyhomologymodeling.TheribbonplotofmatureKLK14isshown
inthetraditionalserineproteasestandardorientation(i.e.lookingintotheactivesitecleft)(86).Secondarystructureelementsaredisplayedasarrows
((cid:3)-strands)andribbons((cid:1)-helices).Thepositionoftheautolysisloopisindicated.N-andC-terminalresiduesareshowninblack;thesidechainsofthe
catalytictriadresiduesareshowningreen;S1sidechainiscoloredorange.KLK14cleavagesites,i.e.P1andP1(cid:1)residues,areshowninredandblue,
respectively,andP1-Rresiduesarelabeled.D,inactivationofKLK14byautodegradation.KLK14wasincubatedaloneat4°C(F),25°C(‚),and37°C(f)
overtime(0–48h).E,SDS-PAGEanalysisofKLK14autodegradationovertimeat4,25,and37°C.Timepointsareindicatedaboveeachlane.M,molecular
massstandardsinkDa.
2412 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
EnzymaticActionofKLK14
intact25-kDabandandacorrespondingdecreaseinenzymatic tive, intact (cid:5)27-kDa band on SDS-PAGE and zymographic
activity(Fig.4,DandE).Auto-inactivationofKLK14byauto- analysesafterprolongedincubation(datanotshown),whichis
lyticcleavagemayrepresentapotentialregulatorymechanism. supportedbypreviousfindings(19).
To test the potential activation of pro-KLK14 by KLK5,
OptimalpHandBufferCompositionforKLK14Activity
whichisoftenco-expressedwithKLK14invivo(32),weincu-
Twobuffersystems,100mMNa HPO (pH7–10)and100mM bated pro-KLK14 with KLK5 and monitored the reaction via
2 4
Tris-HCl(pH6–9),werechosentodeterminetheoptimalpHfor complexformationwithAT.However,wewereunabletopro-
KLK14activity.AlthoughKLK14wasmostactiveatapHof8.0in videevidencefortheactivationofpro-KLK14byKLK5.Neither
both systems, KLK14 activity was 1.4-fold higher in phosphate dose-dependent(datanotshown)nortime-dependentactiva-
versusTrisbuffer.Furthermore,KLK14displayedenhancedactiv- tionofpro-KLK14byKLK5wasobservedinourstudy,ascon-
ity(13%increase)inthepresenceof0.01%Tween20compared firmedbythelackofKLK14-ATcomplexformationafterincu-
with0.2%bovineserumalbumininwhichactivitydecreased30% bation of pro-KLK14 with KLK5 (Fig. 5, lanes 5–9). As
(Tween 20 and bovine serum albumin prevent adsorption of expected, mature KLK14 readily formed a complex with AT,
KLK14). Hence, the buffer composition utilized in subsequent whereaspro-KLK14andKLK5didnot(Fig.5,lanes2–4).Our
studieswas100mMNa HPO ,0.01%Tween20(pH8.0). results are further substantiated by the fact that KLK5 is not
2 4
abletocleaveaheptapeptideencompassingtheactivationsite
ActivationofPro-KLK14
ofKLK14(33)andthatKLK5generallyfavorsArgrelativeto
As with other S1A family SP, pro-KLK14 requires the LysatP1(27),i.e.thebestP1-Argcontainingsubstrate(VPR-
removaloftheinhibitoryN-terminalpro-peptidesequenceat AMC)washydrolyzedata133-foldhighercatalyticefficiency
Lys24–Ile25 for conversion into its enzymatically active form. by KLK5 than the best P1-Lys containing substrate (VLK-
Autoactivation is not expected to occur because P1-Lys resi- AMC)(27).ApriorstudybyBrattsandetal.(19)reportedthat
duesareunfavoredbyKLK14(21)(seebelow).Indeed,purified KLK5 is an efficient activator of pro-KLK14. However, pro-
recombinant pro-KLK14 remained as an enzymatically inac- KLK14activationoccurredafter24hofincubationwithKLK5,
suggestinganinefficientconversionreaction,whichmaynotbe
physiologicallyrelevant.Furthermore,thereactionwasmoni-
toredwithachromogenicsubstrate(Pro-Phe-Arg-p-nitroani-
lide)equallyhydrolyzedbybothKLK5andKLK14.Inourstudy,
weutilizedtheserpinAT,aninhibitorthattargetsKLK14but
not KLK5 (27), to monitor pro-KLK14 activation over 3 h.
Taken together, these results call into question the ability of
KLK5toefficientlyactivatepro-KLK14.
SubstrateSpecificityofKLK14
ThesubstratespecificityofKLK14wasassessedbydetermin-
FIGURE5.Lackofpro-KLK14activationbyKLK5.Pro-KLK14(0.5(cid:2)M)was ing its relative hydrolytic activity and corresponding kinetic
incubatedwithKLK5(0.1(cid:2)M)for0,10,and30minand1and3hat37°C,after parameters (i.e. K and k ) against a panel of 16 synthetic
whichAT,ataKLK14-ATmolarratioof1:2,wasaddedfor30minatroom m cat
temperature(lanes5–9,respectively).Activationofpro-KLK14wasmonitored substrates containing an AMC fluorogenic leaving group
byformationoftheAT-KLK14complex.Ascontrols,ATwasincubatedalone (Table2).AmongtheseAMC-peptides,14containabasicP1
(lane1)andwithmatureKLK14(lane2),pro-KLK14(lane3),andmatureKLK5
residue (Arg, Lys) and 2 contain bulky, hydrophobic amino
(lane4).NotethatanAT-KLK14complexwasnotobservedafterincubationof
pro-KLK14withKLK5,indicatingalackofactivation. acids at P1 (Tyr, Phe) and thereby represent substrates for
TABLE2
Summaryofsteady-stateparametersforhydrolysisofsyntheticsubstratesbyKLK14
Substrate K k k /K Activity
m cat cat m
mM min(cid:2)1 mM(cid:2)1min(cid:2)1 %
Trypsin-like
Boc-QAR-AMC 0.045(cid:7)0.0039 382.07 8516.94 100
Boc-VPR-AMC 0.043(cid:7)0.0067 361.08 8422.67 98.9
H-PFR-AMC 0.090(cid:7)0.018 137.49 1520.57 17.9
Boc-FSR-AMC 0.278(cid:7)0.027 388.68 1397.63 16.4
Boc-LGR-AMC 0.0577(cid:7)0.00680 78.12 1352.96 15.9
Boc-QGR-AMC 0.139(cid:7)0.0241 145.91 1051.98 12.4
Tos-GPR-AMC 0.173(cid:7)0.0187 139.01 803.53 9.4
Boc-QRR-AMC 0.0268(cid:7)0.00284 16.43 612.37 7.2
Benzlyoxycarbonyl-GGR-AMC 0.130(cid:7)0.0146 36.89 282.90 3.3
Boc-LKR-AMC NRa
Boc-LRR-AMC NRa
Boc-VLK-AMC 0.578(cid:7)0.0307 62.23 107.72 1.3
Tos-GPK-AMC NRa
Boc-EKK-AMC NRa
Chymotrypsin-like
Suc-AAPF-AMC NRa
Suc-LLVY-AMC NRa
aNRindicatesnoreaction.
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2413
EnzymaticActionofKLK14
attributedtotheinteractionbetween
AMC and the P1 amino acid (e.g.
cleavageofachymotrypsin-likechro-
mogenic substrate, Ala-Pro-Tyr-p-
nitroanilide,byKLK14wasreported
in a previous study (19)), and 3) the
finalenzymeconcentrationused.
The P2 specificity of KLK14 was
examined by comparing the
k /K values among substrate
cat m
subgroups with invariable P1 and
P3 residues as follows: 1) QAR-
AMC,QGR-AMC,andQRR-AMC;
2) LKR-AMC, LRR-AMC, and
LGR-AMC; and 3) GPR-AMC and
GRR-AMC. As shown in Table 2,
KLK14exertsminimal,ifany,cata-
lyticactivitytowardsubstratescon-
taining basic residues (i.e. Arg and
Lys)atP2andinsteadprefersrela-
tively small, aliphatic amino acids
(i.e. Gly and Ala) at this position.
These results concur with those of
ourphagedisplayedlibraryscreen-
ing, because the vast majority of
FIGURE6.KineticsofinactivationofKLK14by(cid:1)-antiplasmin(A),(cid:1)-antitrypsin(B),antithrombinIII(C),
atinodns(cid:1)o1f-aenatcihchsyemrpointrfyoprsvinar(iDou).sKtLimK1e4i(nfitnearvlcaolsnacfe2tnetrrwathioicnh,0r.e1s(cid:2)idMu)awlaacs1tiinvcituybaagteadinwstitQhAinRc-rAeMasCin(g0.c2omncMenfitnraa-l KtaLinKe1d4P-s2ealemctinedopaceindtsawpeitphtiadleipshcaotnic-
concentration)wasmeasured.A,APfinalconcentration((cid:2)M):0(f),0.1(F),0.2(E),0.4((cid:1)).B,ATfinalconcen- side chains (i.e. Gly, Ala, Val, and
tration((cid:2)M):0(f),0.1(F),0.2(E),0.4((cid:1)),0.6(ƒ).C,ATIIIfinalconcentration((cid:2)M):0(f),0.4(ƒ),0.6(Œ),0.8(E),
1.0(F).D,ACTfinalconcentration((cid:2)M):0((cid:2)),0.5(F),1.0(E),1.5((cid:1)),2.0(ƒ),2.5(f).Theinsetsshowa Leu),whereasnonepossessedbasic
double-reciprocalplotofthepseudo-firstorderrateconstant(k(cid:1))andtheinhibitorconcentration.Theline P2residues(21).
drawnisaleastsquaresfitoftheexperimentalpoints.TheequationofthelineforAPisY(cid:8)42.02X(cid:4)0.32(r(cid:8) TheP3preferenceofKLK14was
0.981);AT,Y(cid:8)20.07X(cid:4)0.12(r(cid:8)0.980);ATIII,Y(cid:8)673.7X(cid:4)3.57(r(cid:8)0.998);ACT,Y(cid:8)5.54X(cid:4)2.0(r(cid:8)0.996).
risthePearsoncorrelationcoefficient. assessed by examining kcat/Km val-
ues in substrate subgroups bearing
TABLE3 thesameP1andP2aminoacidsasfollows:1)VPR-AMCand
InhibitionofKLK14activitybyserpins GPR-AMC;2)LGR-AMC,QGR-AMC,andGGR-AMC;and3)
Serpin KI k(cid:2)2 k(cid:2)2/KI QRR-AMCandLRR-AMC.Yet,unlikeitsrelativelystringent
(cid:2)M min(cid:2)1 (cid:2)M(cid:2)1min(cid:2)1 P1andmodestP2preferences,KLK14possessesalowerspec-
Plasminogenactivatorinhibitor-1 NDa ificity at P3. Specifically, the S3 subsite can accommodate a
(cid:1)-Antitrypsin 0.162 8.098 49.8
(cid:1)1-Antiplasmin 0.130 3.100 23.8 varietyofaminoacids,includingbasicresiduesandthosewith
2
AntithrombinIII 0.189 0.230 1.48 largeandsmallaliphaticchains(21).
(cid:1)-Antichymotrypsin 5.580 1.247 0.224
1
aNDindicatesnotdetermined. RegulationofKLK14Activity
trypsin-likeandchymotrypsin-likeserineproteases,respec- Serpins—Several extracellular inhibitory members of the
tively. In accord with our previous study (21), KLK14 dis- serpinsuperfamily,namely(cid:1)-antitrypsin(AT),(cid:1)-antichy-
1 1
played trypsin-like specificity with a greater catalytic effi- motrypsin (ACT), antithrombin III (ATIII), (cid:1)-antiplasmin
2
ciency for Arg versus Lys at the P1 position, as substrates (AP), and plasminogen activator inhibitor-1 (PAI-1), were
withthehighestk /K valuescontainP1-Arg.TheP1-Arg assayed for their inhibitory capacity against KLK14. Such
cat m
preferenceofKLK14wasfurtherdemonstratedbytheloca- inhibitory serpins behave as irreversible suicide substrate
tion of KLK14 cleavage sites within intact proteins (Fig. 4; inhibitorsandarecleavedbytheirproteasetargetsatascis-
Table 5), all of which occurred C-terminal of Arg residues. sile bond (P1-P1(cid:1)) within their exposed reactive site loops
However,incontrasttopriorfindings(19,21),wecouldnot (RSL), after which the interaction may proceed via the
ascribe chymotryptic-like specificity to KLK14 under the “inhibitory” or “substrate” pathway, resulting in complex
experimental conditions chosen, i.e. no hydrolytic activity formation or an inactive serpin and an active protease,
againsteitherAAPF-AMCorLLVY-AMCwasdetected.Possi- respectively (34). The kinetics of inactivation of KLK14
ble explanations for this finding include the following: 1) the activity in the presence of increasing serpin concentrations
inherent incompatibility of the AMC-substrates used in our aredisplayedgraphicallyinFig.6.Thecorrespondingkinetic
studywithKLK14(i.e.lackofP(cid:1)residuesandthepresenceof constants(K ,k )arelistedinTable3andwerecalculated
I (cid:4)2
unfavored residues at P2 and P3), 2) possible steric effects from the double-reciprocal plots of the pseudo-first order
2414 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007