New insights into the substrate specificity of macrophage elastase MMP-12 Anne-Sophie Lamort, Rodolphe Gravier, Anni Laffitte, Luiz Juliano, Marie-Louise Zani, Thierry Moreau

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Anne-Sophie Lamort, Rodolphe Gravier, Anni Laffitte, Luiz Juliano, Marie-Louise Zani, et al.. New insights into the substrate specificity of macrophage elastase MMP-12. Biological Chemistry, De Gruyter, 2016, 397 (5), pp.469-484. ￿10.1515/hsz-2015-0254￿. ￿hal-01398704￿

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Distributed under a Creative Commons Attribution - ShareAlike| 4.0 International License Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New nal print and online version of the Journal. Any use of these articles s e l c i t r a e s e h t f o e s u y n A . l a n r u o J e h t f o n o i s r e v e n i l n o d n a t n i r p l a fin e h t n i d control quality prior full process advanced to publication. e t c subject the is toexplicitunderstanding through the yet that the not gone papers have e r r o c as published errors articles in r fie i t and typesetting been editing, proofreading, has performed. Therefore, may there be n e d I However, process. of which the additional editorial none includes preparation, copy t c e ‘Just Biol Chem Accepted’ Papers j b O l a t i Disclaimer g i D ’s e l c i t r a the same as DOI e h t journal onthe version d is replaced Web bythe site final n the paper version ofthe with a online issueof , e g a paperin Theproofreading. published its is then edited final p print formregular in and a s t production which process, editing, through the includes normal copy and typesetting n e t n After a in published paper is o C f o e l b a T it in by indicatingauthor form), be articles should and cited list, other title DOI. e h t as published n o d 10.1515/ e t a c (DOI), unique a identifier i for intellectual d property in digital the environment (e.g., n i ‘Just Biol Chem Accepted’ Papers with an grouped papersin issue. other graphics. papers doappearbe When theyfeature print, from will removed in this and theversion final and version. be may There differences inquality also the of editing lead differences or Copy small proofread. to may between the Just Accepted have and buttypeset, manuscript copy are inbeen online published form, notedited, of appearing advance the journal. in print been have They peer-reviewed, accepted Biological ‘Just Chemistry Accepted’ Papers Biological Chemistry ‘Just Accepted’ Papers hsz -2011-xxxx), top shown at is themargin Once an of page. is the article title Biol Chem ‘Just Biol Chem Accepted’ Paper Biol Chem. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Biol Chem ‘Just Biol Chem Accepted’ Paper version. Comment citer cedocument: At this time, the time, At this Biol Chem ‘Just Biol Chem Accepted’ Paper Brought to youby|University ofCalifornia Biol Chem ‘Just Biol Chem Accepted’ Papers Download Date |1/25/16 2:18 AM

are citable; the online are citable; is the publication date have undergone have peer-review the complete Authenticated Biol Chem ‘Just Biol Chem Accepted’ Paper are online,in papers published (and before it (andfinal before in is published its form, proceeds it that will be that will Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 e-mail: [email protected] *Corresponding author b a 2 1 Zani Anne-Sophie Lamort Research Article Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New Current address: Atlanbio,Z.I. de Brais - 1 Current address: INRA UMR1324, CNRS UMR6265, Centre des Sciences du Goût et de l'Alimentation, Escola PaulistadeMedicina,Univers U1100,Equipe 2Mécanism INSERM CEPR UMR Université de Bourgogne, F-21000 Dijon, France France Dijon, F-21000 Bourgogne, de Université Faculté deMédecine,UniversitéTours, 1 andThierry Moreau New insightsintothesubstratespecificityof (5), 469-484. ,DOI :10.1515/hsz-2015-0254 macrophage elastaseMMP-12 1 , RodolpheGravier Comment citer cedocument: 1, * Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

rue Graham Bell,F-4460 Biological Chemistry ité deSaoPaulo,04044-020Brazil Authenticated 1,a 10Bd.Tonnellé,F-37032ToursCedex,France 1 /33 , AnniLaffitte es Protéolytiquesdansl’Inflammation, DOI: 10.1515/hsz-2015-0254 0 Saint Nazaire, France 1,b ISSN (online)1437-4315 , LuizJuliano ’JustAccepted’paper 2 , Marie-Louise Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Keywords: cleavage sitesinitsknownsubstrates.Human synthesized fourseriesoffluorogenicpeptid biological fluidshasbeenhampered bythelack active onnumerous substratesincludingelas (COPD). Thecatalyticdomain ofMMP-12is believed to playa major role inthe developm Macrophage elastase,orMMP-12,ismainly Abstract specificities despitetheir structural similarity. by murine MMP-12suggesting that human and RNALAVERTAS-EDDn andMMP-12residues.Most experiments revealedunexpectedinterac terminal hemopexindomain influencessubs efficiently by full-length MMP-12thanbyits catalytic domainalone, indicatingthat the C- was themost selective substrate for MMP-12ever contrary, thesubstrateAbz-RNALAVERTAS-E substrates compared tootherMMPs, in containing aProatP3inthesequencePro-X-X Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New MMP-12; macrophage elastase;enzyme peptide-protein docking. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: Substrate specificityofMMP-12 Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

cluding MMP-2,MMP-7,MMP-9andMMP-13.In Authenticated 2 /33 e substratesbasedonthesequencesofMMP-12 tions betweenthepeptidesubstrateAbz- tin. However,measuring MMP-12activityin MMP-12 efficientlycleaved unique among MMPsinth ent ofchronicobstructivepulmonary disease producedbyalveolarmacrophages andis DDnp derivedfrom theCXCR5chemokine of highly selective substrates. We therefore trate bindingand/or reported.Allsubstrates ↓ murine MMP-12havedifferentsubstrate Leu butlackedselectivity towards these of oursubstrates specificity;substrate recognition; were poorlycleaved catalysis. Docking werecleaved more at itisquitehighly peptidesubstrates Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 sites ofinflammation(Shapiro Macrophage elastase,orMMP-12,ispredomin Introduction (SNP) intheMMP-12 genearelikelytobe results ofnumerous genetic studiesindicate rather fromtheincreased productionofotherMMPs(Finlay emphysematous lungdoesnotresultfrom an in human, theincreaseinmatrix-degrading patients withCOPD(Demedts evidence that theMMP-12 geneisactivated by the allegedinvolvement ofMMP-12inhuman (Hautamaki mice areresistantto disease (COPD)(Banda a pivotalroleinthepathologyoflungdiseas enhancing theelastinolytic/proteolyticpotential inhibitor (Banda called Studies carriedoutinth and CXCchemokines (Dean proteins (seeref(KaynarandShapir II, andIVcollagens(butnot other MMPs,itcanbreakdownawidevarietyof encoding genewithintheMMP including its secretionas anactivatableprofor much smaller quantities(Lavigne types, includinghuman bronchial derived dendriticcellsalsosynt et al. MMP-12-deficient mice aresignificantly poorer keeping withthishypothesis,Shapiro's grou neutrophil elastase Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New , 1996)thanthoseofwildtypemice. Ina α 1-anti-trypsin)isreadily et al. et al. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 , 1997).However,itisdifficulttoex in vivo lungemphysema inducedbyprolong , 1980).Thisresultsuggestedthat Comment citer cedocument: e early1980sshowedthat et al. andmost MMP-12substratesare associatedwiththeECM.In et al. fibrillar collagens)aswell Substrate specificityofMMP-12 , 1980).Thiswasbecause et al. Brought to youby|University ofCalifornia hesize thisprotease(Kis-Toth et al. epithelial cells, alsosynthe , 2008). et al. Download Date |1/25/16 2:18 AM cleaved by MMP-12, which results in inactivation of the cleaved byMMP-12,whichresultsininactivationofthe

, 2006;Molet o, 2012)forareview)suchaspro-TNF- clusteronchromosome 11(Kaynar , 1993),althoughbothresti , 2004).MMP-12hasmany featurestypicalof MMPs, Authenticated 3 /33 thatseveralsinglenucleotide polymorphisms ddition, they alsoshowed thatthese MMP-12 m, itsdomain structureandthelocationofits ofneutrophilicandmacrophagic cells,played increase inMMP-12synt es especiallychronic proteasesofmacropha p later demonstrated th antly producedbyactivatedmacrophages at cigarette smokingandishighlyexpressedin COPD/emphysema isstilldebated,despite at breakingdownECMsubstrates(Shipley associated withthe prevalence ofCOPD et al. α ECMsubstrates,includ 1-PI( trapolate thesefindingstohumans and , 2005).Otherauthor α 1-PI isthepredominant inhibitor of MMP-12, bydirectlyandindirectly α fibronectin,laminin andnon-ECM -1-proteinase inhibitor, sometimes size andsecreteMMP-12,butin ed exposuretotobaccosmoke et al. et al. ng andactivedmonocyte- , 2013). Some other cell , 2013).Some other , 1997).Similarly, the obstructive pulmonary at macrophages of hesis/secretion, but ing elastin,typesI, gic origininthe et al. s haveshownthat α , plasminogen , 2012).Like -/-

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 not associatedMMP-12genepo (Haq specificity ofMMP-12. was highlyselectiveforhuman Ourre MMP-12. MMP-9 andMMP-13,weidentifiedonesubstrat Although mostsubstrateswere are basedonthesequencesofsitesinMMP-1 developed four seriesof internallyquenched – thoughnotexclusivelyinthepresence currently available are notsufficiently selective for determining MMP-12 activity, especially clefts, whichresults in highly redundant subs other relatedMMPslikeMMP-2andMMP-9, The twomain reasonsare thattheconcentration Selectively measuring MMP-12enzyme activityin toberesolved(Marchant virus-induced inflammation immunity istoproteolyticallyclearinterf of theantiviralmolecule inte as atranscriptionfactor driv antiviral immunity.Theprotease may enterthenu been expanded recently. Indeed,MMP-12 seems to picture ofMMPssecretedasproforms thatareac aortic aneurysm (Curci arthritis (Liu MMP-12 isthoughttobe associated withother contribute toperpetua properties inmice (Houghton and tropo-elastinbyMMP-12(Heinz lung matrix degradation.Forexample, thefrag active biologicalfragments (matrikines) from EC (Schirmer Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. et al. , 2011;Hunninghake et al. , 2009;Zhou (5), 469-484. ,DOI :10.1515/hsz-2015-0254 , 2004;Wang ting lunginflammation. Comment citer cedocument: et al. ing thetranscriptionofI et al. Substrate specificityofMMP-12 rferon-alpha. Thesecond,distinct et al. , 1998),inadditionto readilycleavedbyother Brought to youby|University ofCalifornia et al. , 2013).MMP-12canalso,likemany otherMMPs, generate et al. Download Date |1/25/16 2:18 AM , 2006);theyhelprecruitmonoc lymorphism withthepreval

, 2009;Wallace , 2004),atherosclerosis(Matsumoto et al. Authenticated 4 /33 , 2010;Taddese fluorogenic substrates eron-alpha fromtheblood,soallowing ments resultingfrom th of MMP-2andMMP-9.We havetherefore 2 naturalsubstratescl of MMP-12isoften much lowerthan thatof tivated andthendigestvarioussubstrateshas pathological conditions,suchasrheumatoid M proteins,aswellha trate recognition. Thesyntheticsubstrates e derivedfrom thechemokine CXCL5that cleus ofavirus-infectedcellwhereitserves haveanovel unexpectedroleinregulating sults alsoshednewlightontheenzyme complex biologicalfl that MMPs haveclosely related catalytic itsroleinlungdisease.Theclassical et al. et al. κ B MMPs, includingMMP-2,MMP-7, α , 2014). gene.Thisresults inthe secretion , 2002),whileotherstudieshave et al. role ofMMP-12inantiviral ence orseverityofCOPD ytes tolungtis , 2009)havechemotactic whose peptidesequences eaved bytheprotease. e breakdownofelastin ving adirectrolein uids isachallenge. et al. sue andthus , 1998)and Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 inappropriate forthesmall, syntheticsubstrat the frequency ofall20 amino acidsineach substratefor tworeasons.First, itwas that aProatP3wasoftencorre relatively fewsequencesanalyzed.Howevervisu cleavage sitesgavenosignificant matches be Information, TableS1).Analysisofcorrela substrates and176cleavagesitesin16structur each of74uniquecleavage sitesin44 distinct We determined thefrequencyofeachamino ac including soluble,globularproteinsandthe MMP-12, weperformed oursequenceanalysisontw structural proteinsand/ MMP-12. Becausesomeacids (e.gAla, amino position oneithersideofthecleavagesite.Th substrates resultedinaconsensuspeptidese each amino acidinthe regionsurrounding thecleavage sitesofavarietyMMP-12 (Cobos-Correa MMPs, evenifitwereusedtodesignaFR fluids, whereMMP-12isoftenaccompaniedby substrate maybeunsuitablefordetecting are only3.5-fold(MMP-13) andabout6-fold MMP-13 and MMP-9alsoseem tocleaveitefficiently:their k (Devel sequence wasintroduce and isfrequently usedtomeasure MMP-12activ few ofthem arehighlyselectivefor agi There aremany commerciallyavailablesubstr Designing selectivesubstr Results L-2,3-diaminopropionyl)) asquenchergroup, Dpa-AR withMca(methylcoumarin) asfluor Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. et , 2006).Although thissubstratewasclai et al. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 , 2009), tovisualize itsactivity or proteinsof theECMthat are Comment citer cedocument: d (Mca-PLGLEEA-Dpa-NH ates forhumanMMP-12 Substrate specificityofMMP-12 lated with a Leu at P1’.Welated withaLeuat ma Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

Authenticated ven MMP.Thefluorogenic 5 /33 and quantifyingMMP-12activityinbiological ET probesupposedtobeselectiveforMMP-12 es includedinourstudy solubleMMP-12substrates,includingsynthetic other comprising structuralandECMproteins. escent groupandDpa(N ted mutations inthe regions surroundingthe was originallyde quence thatretained key amino acidsateach (MMP-9) lessactivethanMMP-12.Thusthis ates formost MMPs,includingMMP-12,but al substrateslikeelas id from positionP4 to P4’(Figure 1) within ity. Itwaslater modifi is peptideshouldbeselectivelycleavedby higher concentrations ofMMP-9andother tween residues, possiblybecauseofthe al inspectionofthesesequencesrevealed in vivo Gly, Val,Pro)areover-represented in o differentsetsofproteinsubstrates,one 2 ) toenhance selectivity for MMP-12 substrates forseveralMMPsincluding med tobe selectiveforMMP-12, . Ouranalysisof de nocorrectionstoaccountfor cat signed byKnight / K m valuesindicate thatthey tin (seeSupplementary and secondbecausewe ed andaGlu-Glu(EE) -3-(2,4-dinitrophenyl)- substrate Mca-PLGL- the occurrenceof et al. (1992) Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 α α (Gronski Phe 352-Leu353scissilebondinhuman PP-17 substratecontainsbothaPhe-Leuand cleavage sitesinproteinsreportedtobephys The lastseriesoffluorogenicsubstrates (Table 1). whichwasthenmodifiedtogi RPLGLGGAQ, insoluble substrates.Thisgeneratedth takes into accounttheamino acidsmost fr The thirdseries offluorogenicsubstrates(Group III)isbased onaconsensussequence that generate asubstratecleavedonlybyMMP-12(Table 1). hydrolysed byMMP-9andMMP-13withsimilarly synthesize PP-2b.Thissequence,whichissaid The second substrateseries (Group II)is residue inPP-23 inordertoassessthe charged arginine atP4ons residue, glutamate, atposition P4in PP-21substr structure-function information synthesising substratesmodified atdifferent Abz-RPLALWRSQ-EDDnp (PJ-1)wassynthesizedand usedastheleadsubstrate for hence wasunlikelytoprovideaselective (Figure 1). Thislattersequence wasnotexploi andanalysisofthe«insolubl RPLALWRS amino-acid sequencederivedfromtheMMP-1 (Banda becoming mostsusceptible, probablyas These analyses ofsoluble substrates gave regions oftheproteinsubstrate. assumed thatthecleavagesiteisrecognizedas Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New 1-PI ledto ashift in themouse 1-PI cleavedbymouse MMP-12(Banda et al. et al. , 1987).Forothersubstrates ofthis , 1997),whilethelattercorresponds (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: ubstrate hydrolysis. We alsoreplacedthe ProatP3byaGly Substrate specificityofMMP-12 Brought to youby|University ofCalifornia (Table 1). Forexample, weintroduced anegatively charged Download Date |1/25/16 2:18 AM MMP-12 cleavagesite, withthePhe 352

importance ofprolineatthatposition. Authenticated tested wasbasedonsequencesspanningMMP-12 α based ontheleadsequencePLGLEEAusedto 6 /33 substrate forMMP-12.Thefluorogenicpeptide et al. e PP-6substratecont 1-PI cleavedpreferentiallybyhuman MMP-12 us theconsensussubs equent ateach positionin bothsoluble and ted because ithad lowcomplexity content and positions toincrease selectivity ortoobtain aconsequenceoftheoxidationMet358 iological substrates of MMP-12. The 13-mer iological substratesofMMP-12.The13-mer e »onesgavethesequenceGPAGLGGA Pro-Met bond.Theformer correspondstothe to beefficientlycleavedbyMMP-12,isalso suchregardlessoftheamino acidsinother 2 cleavagesitesintwo CXCchemokines , 1987).Itisnoteworthy ve twoadditionalPP-6 series (PP-14,PP-15,PP-16), weusedthe ate toevaluate theeffect of thepositively to thePro 357-Met358bondinhuman activity. Itwasoptimized toattempt to trate peptidesequence aining thesequence -derived substrates that oxidationof

– Leu353bond

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 may affect concentration. sequence, possiblyresultinginth extinction coefficientcouldbedifferentfor spectrophotometrically usingthemolar extin substrates andattribute them tothefact directly (firstorderconditions).We haveprevi calculated fitting data totheMichaelis-Menten equationus first-order kinetics (S<< fluorescence measurements usingalowsubstrat We first determined the Characterization offluorogenic subs C-terminus. ThekineticsoftheirMMP- peptides bearinganAbz fluorescentgroupat All 21oftheabovepeptidesweresynthesize MMP-12 substrate. Hence peptidesequencesbasedontheseproteoly specific sites (Dean,Cox chemokines may beactivatedorinactivated (Dean, Cox k calculated from individualparameters For some substrates, there werediscrepa (Table 2). only forthose substratesthatwereefficiently cleaved as assessed by their above 20µM(Korkmaz proportional totheincreaseofpeptideconcentr inner filtereffectathighconcentrations as determined from active-sitetitration.Becau Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New cat / K m valuewhichdependsonlyontheenzyme notonsubstrate concentration, k K cat et al. m from theequation determination using theMichaelis-Menten (5), 469-484. ,DOI :10.1515/hsz-2015-0254 2008)onesitein Comment citer cedocument: K et al. m et al. k ). Theindividualparameters cat Substrate specificityofMMP-12 / K , 2008).Hence,wedetermined 2008)thataredifferentfromthoseattackedbyotherMMPs. Brought to youby|University ofCalifornia m V e under-orover-estim ratiosforsubstratehydrolysisbyMMP-12fromdirect Download Date |1/25/16 2:18 AM m

= CXCL1 andtwositesinCXCL5.AlthoughELR k cat k 12-mediated cleavage wereanalyzed. cat x[E] Authenticated trates forhumanandmurineMMP-12 ncies ofdifferingim and 7 /33 leading toadecreaseoffluorescenceyield the wholesubstrate,dependingonitspeptide that substrateconcentration wasdetermined t ction coefficient of the EDDnp group. This ction coefficientoftheEDDnpgroup. by various MMPs,MMP-12seems totarget se Abz-peptidyl-EDDnpsubstratesexhibitan ation, wedidnotusesu K where [E] ously seensuchdifferencesforthistypeof d asshortinternallyquenchedfluorescent ing non-linearregression analysis. Wethen m theN-terminus andanEDDnpatthe e concentrationsothathydrolysisobeyed tic sitesmay beusedtodesignaselective andthosesame ation ofsubstrateconcentrations.This K t fitbutnotthedi m is theactiveenzyme concentration and portance betweenthe individual kineticparameters V m werealsodetermined by k cat / bstrate concentrations K m ratiosdetermined rectly determined k cat / K m + k value CXC cat / K m

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 highest (80mM value forPJ-1hydrolysis byh-CAT-MMP-12(c was cleaved efficiently byfull-lengthMMP-12with a amino acidsequencesgeneratedthebestsubstrat Remarkably, ourattempts to identify sensi PP-16 provedtobeveryselectiv cleavage sitesfrom theAla-Leu C-terminal sideofthescissileAla-Leu bondin major cleavagesitewasstillattheAla- Replacing Pro withGlyinthesubstr catalysis atthepeptidebondwheresecondLe cleaved, suggesting thattheProresidue influences contained asecondLeuinthemotif Pro-Le strong preferenceofMMP-12forthisresidue human MMP-12atthepeptidebondN-terminal to variants (groupI),werecleave All thesubstrates inthe first series contai substrates forhuman interms MMP-12 of MMP-12, human substrate series comprising sequences spanning s the firstthreeseries,exceptPP-23,werehydrolysedwith MMP-12 ismuchmore efficientat 2009) withoutthecontributionofhemopexi catalytic domain alonecan than those for thecatalytic domain alone. Thisresult confirms early reports that the MMP-12 MMP-12 weresignificantly greater (6-times fo appeared tobecatalyticallyactive,the domain andthefull-length instead. We detectedno differe MMP-12 foraLeuatP1’;theGly-LeuorGl at theappropriatebond.ThePro-Leubondwasnot series ofsubstratesconfirmed theimportanceof Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New -1 . PP-23appearstobearather -1 (5), 469-484. ,DOI :10.1515/hsz-2015-0254 s α -1 1-PI, human CXCL1andmurine ) among allthesubstratestested.Modifying thePJ-1sequenceby Comment citer cedocument: hydrolyseelastin(Curci forms ofhuman MMP-12.Altho Substrate specificityofMMP-12 Brought to youby|University ofCalifornia poorsubstrateforbothformsofhuman MMP-12.The fourth d byboththecatalyticdomain e forhuman MMP-12(seebelow). nces betweenthelocationsof or Glu-Leubonds(PJ-5).The Download Date |1/25/16 2:18 AM cleaving substrates, even small ones.All thesubstrates in

ate PP-23dramatically decreased the k cat Authenticated Leu bond.Also,modifying thesequenceon / k tive substrates for MMP-12 based on consensus tive substratesforMMP-12basedonconsensus K 8 /33 ning the lead consensus sequence PJ-1 and its ning theleadconsensussequencePJ-1andits cat m / valuesforsubstrate r PP-20andPP-6upto35-times forPJ-3) u, but this Pro-Leu bond was almost never u, butthisPro-Leubondwasalmostnever K PJ-1-derived substratesdidnotalterthemajor u-Leu bondswerealmost invariablycleaved theProatP3 positionfordirectingcleavage es inourfourgroups.TheleadsequencePJ-1 n-like domain. Italsoshowsthatfull-length n-like domain. atalytic domainofhum m thepeptide conformation tofoster efficient comparedtogroupI,IIandIII;however thecentralleucineresidue,confirming the at P1’.AllthesubstratesexceptPP-23, cleavage sites innatural substrates of u occupiestheS1’su et al. cleaved despitethemarked preferenceof k cat , 1998)andcollagen(Taddese k / K cat CXCL5, provided lessefficient CXCL5, provided ugh theMMP-12catalyticdomain m / K ratioof920mM m sites cleaved bythe catalytic valuesofabout4to80mM data forthesecondandthird andthefull-lengthform of hydrolysis byfull-length an MMP-12)wasthe bsite oftheenzyme. k cat / K -1 m s althoughthe -1 , whilethe et al. -1 ,

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 domain alone binding and/orcatalysis ofour histidine tag (6×His) of thecatalytic dom significantly more activethanthecatalyticdomai The Human full-length MMP-12 ha 145 mM at P4inPP-2b increased the better thanPP-2b,thesubstrate regardless of cleavageselectivity. PJ-8 is15 substrates PJ-8andPJ-1appe that theyhavedifferentreporter-que MMP-12 that iscleaved witha is almost identicaltothat forMca-PLGLEEA-Dpa-NH contains thePLGLEEAseque k efficiency. TheGlu-GluinPJ-8providedth introducing ArgatP2’(PJ-4)orGluP1 representative substrates ineachseries degradation patterns were slightly murine MMP-12.Cleavageisprobablynotatthe same peptidebondbecausetheSDS-PAGE natural substrateofMMP-12,is This issupportedbytheobservationthathuman that thesubstratespecificityof biological fluids,provid full-length mouseMMP-12thanbyhuman MMP-12. TheexceptionwasPP-22,whichcl Surprisingly, most ofthem werepoororvery substrates withmouse MMP-12,eitherthecata Because themouse isanappropriatemodel fo is important fordefining first, PJ-1-derived,seriesalso Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New cat / K k m cat of1600mM / K -1 m s valuesforhydrolysisofour21substrat -1 ) may beusefulforevaluatingthe (5), 469-484. ,DOI :10.1515/hsz-2015-0254 -1 s Comment citer cedocument: ed itispresentasafull-length -1 substrate specificity. . Thisisabout2-times greater Substrate specificityofMMP-12 nce, wascleavedwitha k ar tobethebestsubstrates mouse MMP-12may differfrom had anArgatP4,suggestingthat substrates, wedetermined cat Brought to youby|University ofCalifornia containing thePLGLEEAsequence. k / cat different(datanotshown). K cleaved initsinhibitoryr Download Date |1/25/16 2:18 AM / m

s abettercatalyticeffici K ofPP-2hydrolysis6-fold. m of185mM ncher pairs(Abz-EDDnp andMca-Dpa-NH by commercial nontaggedcatalyticdomain ofhuman Authenticated FL-MMP-12 (Table2).However,PP-22( 9 /33 ain ofMMP-12may haveaninfluenceon the -times betterthanPP-2bandPJ-1is8-times e bestsubstrateforhumanMMP-12,witha (PJ-5)did notsignificantly altercleavage lytic domain aloneorth r studyinglung diseases, wealsotestedour poorsubstratesofbothforms ofmouse n alone.Toruleoutthepossibilitythat α -1 1-PI, aproteinaseinhibitorsaidtobe s activity ofmouse MMP-12inmurine es indicate that full-length MMP-12 is k eaved 4-times less -1 cat (Devel 2 , awidelyusedsubstrateforhuman / form enzyme. Theseresultssuggest K everreportedforhuman MMP-12 k than thatforPJ-1.PP-2b,which m cat eactive loopbybothhuman and ratioof110mM / K ency thanthecatalytic m theS4subsiteofenzyme that ofitshuman counterpart. et al. valuesforthehydrolysis of Most goodsubstratesinthe InsertinganArg residue , 2006), despite the fact , 2006),despitethefact e full-lengthenzyme. efficiently bythe -1 s -1 . Thisvalue 2 k ). Our cat / K m : Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 h-CAT-MMP-12, K efficiency ( analysis toevaluatetheeffectsoftargetedamino acidchanges insome substratesoncatalytic MMP-9 andMMP-13)that arelikel determined the X All thesubstratesingr Selectivity ofMMP-12 substrates The h-FL-MMP-12 compared toh-CAT-MMP-12 ispr h-CAT-MMP-12 indicate bindingaffinity.Therelative maximum substrate substrate affinitythantheLeuatP1'andPro- deleterious influenceonsubstrat Glu withabulkyhydrophobicTrpresidueor which hasjustanadditionalArg resulted inthelowest influence onthebindingaffinityofsubs not greatlyaltersubstr Thus, amino acidchanges did notvarysignificantlybetweenbothforms of of the Then wedetermined individualkineticpara interfere withtheenzyme activity suggests thattheC-terminal His-tag,located PP-6 andPP-16bynontaggedCAT-MMP-12 MMP-12. Wefoundnosignificative Most groupIsubstrates were by yetunknownmechanisms. domain ofMMP-12mayhelptoproperlyposition magnitude. Thissuggeststhatevenforsmall- higher (10to25times) thanthoseforthecatal Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New HY isahydrophobicresidue(e.gleucine)that k cat valuesforothersubstrateshydrolyzed K m value(about4-times lower)wasobser k cat ) orsubstrateaffinity( k cat (5), 469-484. ,DOI :10.1515/hsz-2015-0254 / K m valueswerebetween5µMand30 m K valuesfortheirhydrolysis by several MMPs(e.g:MMP-2, MMP-7, oups I,IIandIII,ex Comment citer cedocument: m ate affinity andthe C-terminal valueswithh-FL-MMP-12,althou that thegreater within PJ-1 andPP-2b Substrate specificityofMMP-12 bettersubstratesforMMP- Brought to youby|University ofCalifornia e affinity. Perhaps re Perhaps e affinity. of theMMP-12catalyticdomain. Download Date |1/25/16 2:18 AM residue onitsN-terminal side

y tobeco-localizedwith MMP differencesforthehydrolysisofPJ-1,PJ-8,PP-2, PP-2b, K m ) (Table3).ExceptPJ-8 forwhichamarked decrease Authenticated 10 /33 cept PP-23,haveaPro-X-X- k oppositetotheproteaseactivesitedoesnot cat sized syntheticsubstrates,thehemopexin-like trate. TheGlu-Glumotif inPJ-8 andPP-2b Leu atP3-P2,whichseemtobeessentialfor meters usingclassical / ytic domain, i.eapproximately oneorderof byfull-lengthMMP-12weresignificantly K peptide leadsequences catalyticefficienciesof h-FL-MMP-12and is recognizedbyanumberofMMPs.We a positivelychargedArgresiduehadno thehuman enzyme foragivensubstrate. the substrateforoptimal catalysis,however m edominantly driven by anincrease in versus His-taggedCAT-MMP-12.This for agivensubstratehydrolysedwith sidues P2' andP3' are ved forh-FL-MMP-12compared to µM dependingonthesubstrate,but hemopexin-like domain haslittle gh thiswasnotth 12 thanthey were for MMP-2, (Table3).Replacingeither -12 intissues non-linearregression of most substratesdo ↓ -X less important for HY e caseforPP-2, motif, where like thelung. k cat . Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 140 mM do notcontainthePro-X-X- group IVsubstrates,whosesequencesspanMMP- selectivity ratioforPJ-10wa III substrates more activelythandidMMP-12. Forexample, theMMP-12/MMP-13 with MMP-2andMMP-9. Onceagain,MMP-13catal structural information on the wayresidueson MMP-12 complexedwith inhibitors complexed withuncleaved peptide substratesis these residues contribute tosubstratespeci It isnotclear justhowsubstrates interact Modeling peptidesubstrate evaluating MMP-12proteolyticactivityin Altogether ourresultssuggestthatPP-16 could MMP-1, -3,-8and-14.Asexpected,they finding thatPP-16isaselectivesubstratefo selectivity ratiogreaterthan70,i.ereaching ne unlike thislatersubstrate,itwasverypoorly 21 substratesthatwehavedesigned.It two sitesinCXCL5cleavedbyhumanMMP-12,app a selectivityratioof However, MMP-13cleavedthese substrates more efficiently thandid MMP-2, -7and -9with and PJ-9,werebetterforMMP-12.PP-6a MMP-13. Thesituationwassimilar forgroupsII PJ-8, thebestMMP-12substrateingroupI, ratio: 0.12)andPP-20(selectivity MMP-12 (PJ-1 andPP-21),butwassignificantly much more activeonthesesubstrates.MMP-13 approximately 2(PJ-4withMMP-2)to460(P MMP-7 orMMP-9,withselectivity ratios( significantly PP-16substrates( the same rangeasthosedetermined forMMP-2,-7,-9and-13buttheydidnotcleave Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New -1 s -1 , avaluesimilar tothat (5), 469-484. ,DOI :10.1515/hsz-2015-0254 0.2 forPP-15.PP-16,whosesequenceis Comment citer cedocument: Substrate specificityofMMP-12 s 0.4,thatforPJ-7was0.34andPJ-90.25.Most ↓ k -X Brought to youby|University ofCalifornia interactions withMMP-12 cat HY / Download Date |1/25/16 2:18 AM K ratio:0.3),i.e8-times and3-

motif,werepoorlyhydroly oradductsresultingfrom cl m forMca-PLGLEEA-Dpa-NH < 2mM washydrolyzedby Authenticated nd PJ-9 hadslightlyhigherorsimilarnd PJ-9 wereallabletocleavePJ-9with biological fluidscont with residuesoftheMMP-12activesiteandhow 11 /33 r human MMP-12, wehaveextendedourstudyto r human MMP-12, k ficity becausenoX-raystructureofMMP-12 cat -1 was only5-times betterforMMP-12than hydrolyzed bytheotherMMPstested,witha s arly twoorders ofmagnitude. Toconfirm our / K make anappropriatesubstrateforselectively J-1 withMMP-9).MMP-13appearedtobe -1 and IIIsubstrates,allofwhich,exceptPP-6 available.However,the 3Dstructuresof m ). either the P sideor 12 cleavagesitesinna withMMP-12/ more efficienttowards PP-22(selectivity hydrolysed some ofthemasactively eared tobethemost selectiveofallthe ysed thecleavage h-FL-MMP-12 witha derived from thatofonethe aining otherMMPs. eavage ofsubstratesprovide zed byMMP-2,-7and-9. times betterthanMMP-12. 2 (Devel k cat the P' sideofMMP-12 / of some groupIIand K tural substratesand m et al. withMMP-x)of k cat k / , 2006).But, K cat m / K valuesin k cat m values / K m of Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 determining specificityandsele involved insubstratespecific peptide-enzyme interactions.Thesetwoloops specificity loop(Ala234-Asp244)ofMMP-12 are experiments revealedthatth residues arecloseenoughtothe interactions involvingcharged formed a total ofsevenhydrogenbonds(Fig. binding totheMMP-12activesite,substr (Kridel a relatedenzyme thatalsofavors Argatth but suchanArg-PheinteractionbetweentheAr driving substratebindingmode. Theseresults 171 andPhe185.Ourresultssuggestthatboth bend inthepeptide that enabled the side-chain at docking procedure(datanotshown).Theconformati containing ArgatP4,Pr P’1. Thesedockingresultsallconvergedonthesa binding toensurethatcleavageoccursatthe residues make major contributi interactions. Thisfavorablebi Phe 171(contactarea57.7Å feature revealedbythepeptidedockingisthat hydrophobic contactswithcontac Leu atP2issandwichedbetweentheen would behighlyfavorableatthatposition.Astri completely, suggestingthatalongerside-chain The side-chain of theP1' residueValpenetrat and forms numerous contacts,includinghydrog The peptidesubstratefitssnuglyintothecr submitted thecomplex toFlexPepDockwithout RNALA Ala andGlu represent P1', P2' andP3' residue substrates interact withtheenzyme.Based Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. ↓ VERTAS oftheselective PP-16substrate into theMMP-12catalytic cleft and , 2002).Whilehydrophobicinteractionsma (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: o atP3andLeuP2(e.gPJ-8 2 Substrate specificityofMMP-12 ) andPhe185(contactarea35.5Å nding ofbothLeuP2andArgP5 e residuesoftheso-called residues oftheRNALAVERTASpeptidebecause nocharged Brought to youby|University ofCalifornia ity. Ourresultsreinforce the ctivity forsubstratebinding. ons tosubstratebindingandc active sitecrevice t areasaslarge35.5Å Download Date |1/25/16 2:18 AM

Authenticated on theMMP-12/Ile-Ala-Glu complex, whereIle, 12 /33 evice runningacrosstheactivesiteofMMP-12 Ala-Val bondratherthanatthewithLeu zyme residuesHis172andIle180toform s respectively, wehavepositioned thepeptide of ourdockingexperimentswereunexpected ate residuesAsn2,Va es intothedeep S1' pocketwithout filling it g atP4ofasubstratecomplexedtoMMP-14, differfromoneMMP toanotherandare the ArgatP5interactsstronglywithenzyme any constraintsduringthe dockingprocedure. likethatofleucineorpossiblynorleucine king featureofthedocke at position,hasbeenpreviouslyreported P4tointeract with the enzyme residues Phe en bondswithenzyme residues(Figure2). Phe residuesinMMP-12areessentialfor where peptidesubstratesbind.Ourdocking me featureswhenothersubstrates binding involved inasignificantproportionofthe 2C). We detectedno on oftheProatP3inPJ-8introduceda ke amajor contributiontopeptide 2 and46.5Å S-loop (Ala 167-Leu181)and substrate) wereincluded inthe ould beanchorpointsdriving suggeststhatthesetwo PP-16 2 role ofthesetwoloops in ), probablythroughpi-cation l 6,Glu7andArg8 2 . Anotherinteresting d peptideisthatthe true electrostatic Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 most selectiveforMMP-12, perhaps because less efficiently,byMMP-2,-7,-9and-13.The ex selectivity forMMP-12.Allbut modified itby amino acidsubstitutions togenerate substrates withbetter specificity and the cleavagesitesinsoluble,globularprotei MMPs. We designedaleadsubstrate(PJ-1) a lowcomplexity contentofamino acidan (Taddese MMP-12, whilePro/HyPpredominates atP3a frequent aminoacidat theP4,P1andP3'site also frequentlyfoundatsome positionsinthe contain highproportionsofglycineandpro proteases. Collagen andelastin,twonatural but thisseems tobe less important forMMPsthanotherenzymes, suchasserine not takeintoaccountanyinte sequence preferentiallyrec found ateachpositionfrom P4toP4' onbothsides designing new, selectivesubstratesfor theenzyme comprising bothglobularandstruct We examined sequencesspanningthecleavage PP-23, whichlacksaPro atP3,was apoor (Chen provide quiteahighdegreeof However, insome cases,ithas beenpossible toidentify discriminatory sequences that results indicatethatthesubstraterecogni has beenfrequentlyusedtogenerateverygood Proline isoftenthepreferred residueat longhydrophobicaccommodate side-chains, frequen ion andaglutamic acidresidueinvolvedin The catalytic cleftsofallMMPs have very simila Discussion (where X Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. HY et al. , 2002,2003;Deng isLeuinourstudy)that occursfreque , 2010).Aconsensussequence (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: ognized byMMP-12.This‘amino Substrate specificityofMMP-12 rdependence ofsubsitesfora et al. Brought to youby|University ofCalifornia one ofthePJ-1-derivedsubstr Download Date |1/25/16 2:18 AM selectivity for substrates

, 2000;Kridel ural proteins, in order to identify sequences useful for ural proteins,inordertoidentifysequencesusefulfor substratepositionP3 andthePro-X-X- Authenticated 13 /33 tion profiles of MMPs overlapconsiderably. tion profilesofMMPs line (orhydroxyproline) ECM substratesofMMP-12andotherMMPs, ns thatwererecognizedbyMMP-12andthen using onlythoseprefer all theothers contai derived from theseECM d wouldprobablybe s oftypesIandIIIskincollagens cleavedby substrates forMMP-12 and theotherMMPs sitesinalltheknown substratesforawiderangeofMMPs.These cleavage sites.Forexample,Glyisthemost catalysis, plusadeepS1'pocketthatcan nd Leuisthepreferredamino acidatP1' r structural features.They allcontain azinc . We identified there et al. ception, PJ-8,appearedtobethesubstrate ntly insyntheticsubstrates forMMPs. of thecleavagesite tly aleucine,atsubstrate positionP1'. , 2001). ccommodating substrateresidues, ofsome individualMMPs ates werehydrolyzed,moreor acid consensus’strategydoes n themotif Pro-X-X- cleaved bymany other red amino acidsaround substrates ofMMP-12, residues andtheseare substrates wouldhave sidue most frequently so astogeneratea ↓ -X HY motif motif ↓ -X HY

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Although the the chemokine CXCL5,wasthemost selective substrates. ThesubstratePP-16,withthepe poorer substratesforotherMMPscompared toMMP-12,thanwerethegroupsI,IIandIII derived from proteinsreportedtobephysiolo between substrateresiduesandagivenMMPac side-chains likethoseofAlaandGlyareunlik by MMP-12didnothelpgenerateefficientan The information inferred from analysisof sites that substratesareselective forMMP- favoured insubstratesthat ar Surprisingly, substratePP-16 doesnothaveapr poorly ornothydrolyzed,byotherMMPsincludi selective forMMP-12,PP-16substrateprovedto be widely used asacommercialsubstrate todete binding energy(Gronski,Martin favorably withthedeepS1' pocketasprevious smaller contributiontosubstratebindingener are bounddifferentlyatthesubstrate-enzyme significant effectonsubstratehydrolysis.This positions (P2', P3') of oursubstrates, suchasTrp-->Arg,Trp-->Glu and Arg-->Glu,hadno selectivity forMMP-12 hydrolysis. Surprisingly, Hence onecandeducethatintroducingArgatP4 replacing thisArgwithGluinPP-21hadnode PP-2b). ButotherMMPsalsocleavedtheseP4 PP-2, hadanArgatP4andweremorereadily substrate wassignificantly decreased. Themo cleavage attheAla-Leubond.However,catal also shiftedwithasecond,minor cleavage ( correct positioningofth tested, providingfurtherevidencethataProatP3 Mca-PLGLEEA-Dpa-NH range, itissimilar to thatfortheseque Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New k cat / (5), 469-484. ,DOI :10.1515/hsz-2015-0254 K m for itshydrolysisbyhuman for Comment citer cedocument: e substratefor efficienthydrolysis 2 (Devel e commontomost MMPs(Ratnikov Substrate specificityofMMP-12 Brought to youby|University ofCalifornia et al. et al. Download Date |1/25/16 2:18 AM

1997). , 2006).Butunlike Authenticated 12, andpossiblyforother 14 /33 ptide sequenceRNALAVERTASderivedfrom st suitableMMP-12substrates,likePP-19and rmine MMP-12activitybut maybebecausetheseamino acidside-chains leterious effectsonit in ECMproteins(group ly reportedandprobablyprovidesmost ofthe ≈ gy thandoesP1', where Leuinteractsvery gical substratesof nce PLGLEEA (PP-2b) and to that for nce PLGLEEA(PP-2b)andtothatfor d/or selectivesubstr interface or becausetheseresidues make a oline atpositionP3,a for MMP-12ofallthe21substratestested. 5%)attheTrp-Arg bond,inadditionto isimportant forsubstratebindingand/orthe ely toprovideenoughspecificinteractions cleaved thanthosethatdidnot(PP-20and Arg-containing substratesmore readilyand drastic amino acidsubstitutions atprimed ng MMP-1, -2,-3,-7,-8,-9,-13and-14. tive site.Incontrast MMP-12 doesnotexceedthe10 amuch bettersubstratesinceitwasvery ytic efficiencyof ofsubstratesisnotsufficienttogain Mca-PLGLEEA-Dpa-NH . ThecleavagesiteinPP-23was et al. MMP-12, weregenerally s hydrolysisbyMMP-12. ates for MMP-12. Short ates forMMP-12. MMPs, may beobtained lthough thisresidueis , groupIVsubstrates MMP-12 againstthis III substrates)cleaved , 2014).Thisimplies is clearlynotvery 2 5 which is M -1 s -1

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 state isstabilized andso increase substrateturn-over ( Consequently thismay theninfluencesubstrat resulting inhighconformational freedom interdomain region inMMP-12,i.ebetweenthe efficiently thanthecatalyticdomain alone. This how full-lengthMMP-12cancatalysethehydrolys like insertsofMMP-2andMMP-9forbindingto situation amongMMPs,whichusuallyrequireone elastin (Shapiro alone, althoughthelattermay beactiveenoughtodi Full-length MMP-12,eitherhuman ormurine, is behave similarly. Itcouldalsobeduetothe MMP-13 has ahigher intrinsic activitythanother MMPs. Some serineprotease families substrate aresomewhere between thetwo(Neumann MMP-12 (Palmier NH2), whichisatentimes bettersubstrateforMMP-13(Neumann similar findings, especially withthe comm including MMP-12,althoughthiswasnotthema MMP-13 cleavedmost ofoursubstrates,excep tended tobehighlyfavora important forbothefficienthydrolysisandselect gave aselectiveone(Kridel MMP-14 substrates; replacing thePro atP3inan using sequenceswithoutaprolin activities of FL-MMP-12 and its to thecatalyticdomain. Thustherearereally forms, although therewerealsominor protei (Figure S1)indicatethat full-length proforms accordi examined themolecular forms ofhuman andmurineMMP-12 followingtheactivationof catalytic domain isthe activeform of MMP-12 domain is removed auto-catalytically (Bertini like domain. MMP-12,unlikeotherMMPs,tendsto Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. et (5), 469-484. ,DOI :10.1515/hsz-2015-0254 et al. , 1993)andcollagenV(Fu Comment citer cedocument: most ofthehuman andmurineMM , 2007);theactivities ble inMMP-12substrates. Substrate specificityofMMP-12 ng tothesupplier’sprotocol et al. Brought to youby|University ofCalifornia catalytic domain, both e atP3orotherpositions.Si Download Date |1/25/16 2:18 AM

, 2002).Thisstudyalsoshowed Authenticated with respect toeach other(Bertini 15 /33 on MMPsubstrateFS-6(Mca-KPLGL-Dpa-AR- peptide sequencesofthesyntheticsubstrates. n bandsofabout20kDa, probablycorresponding t PP-16,much moreactivelythanotherMMPs, significant differencesbetween thecatalytic e bindinginsome wayso catalytic domainandthehemopexin domain, of MMP-2,MMP-7andMMP-9withthis et al. ivity. We alsofoundthatanarginineatP4 may beduetothehighflexibilityof jor goalofourstudy.Othershavereported elastin or collagen. Itisnotclear,however, unselectivesubstrate far more activethanthecatalyticdomain in vivo et al. or more exosites,suchasthefibronectin- be processedsothatitsC-terhemopexin gest protease-resistant is ofsmall syntheticsubstratesmore k , 2008),suggesting cat et al. active site-titrated before determining , 2001).Thisseems tobeaunique ) inthepresenceofhemopexin- (Shapiro P-12s mature totheir full-length , 2004).This could bebecause milar resultswereobtainedfor s. OurSDS-PAGEstudies et al. thattheArgatP4as et al. with anotherresidue thatthetransition , 1993). We have , 1993).We have substrates suchas that the22kDa , 2004)thanfor et al. , 2008). Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 emphysema (Hautamaki MMP-12-deficient mice are,unlike normal mice, thought toplayakeypathogenic role.Thisa true roleofMMP-12inprotease-drivenlungdi The differing substratespecifici human MMP-12arepoorlyrec MMP toanother.Thismay provideastructural specificity loop;twoloopsthatareinvolved of MMP-12showedthatmany ofthem (datanot identical, so35%ofresiduesdiffer.Mappingth quite similar. Thesequences of thecatalytic the activesitesubsites(Kalupov elastase arenothydrolyzedbyitsmurineorthol observed asimilar situationwherebyfluorogenic substrates ofhuman arenotreco MMP-12 activity. Thissuggeststhatsequenc MMP-12, andeventhatof collagens, Bhaskaran kinetic constants. Using self-assembling triple helical peptide substrates as mimics of peptide Mca-PLGLEEA-Dpa-NH MMP-12 activitybyitsmanufact otherhand,thes (data notshown).Onthe elastin. As expected, thecatalytic domain act inactive; ourSDS-PAGEandzymography da MMP-12. Thecatalyticdomain andfull-length MMP-12 whenassayedwithoursubstrates, It isnot atall clearwhy thecatalytic activity of mouse MMP-12islower than that of human activity of this enzyme difference inthecatalyticpotentialoftw elements and thecatalytic site.Furtherstudies non-helical. Perhapstherearelong-rangecommuni domain influencedthehydrolysisofourpep values forhydrolysisofthese15-mer substrat Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New (5), 469-484. ,DOI :10.1515/hsz-2015-0254 et al. in vivo Comment citer cedocument: et al. MMP-9,clearlydecrease (2008)showedthattheC-terminal domain hemopexin-like of Substrate specificityofMMP-12 . , 1997).Buttherelevance of ognized bymurine MMP-12. Brought to youby|University ofCalifornia ties ofhuman andmouse MMP- et al. urers, Mca-KPLGL-Dpa-AR,issomewhat differentfrom the 2 Download Date |1/25/16 2:18 AM usuallyrecommendedformeasuring human MMP-12

es deducedfrom analysisof , 2009), although the sequences of the two proteases are , 2009),althoughthesequencesoftwoproteasesare Authenticated 16 /33 domains ofhumanandmurine MMP-12are65% ssumption waslargelybased onthefindingthat ubstrate recommended determining for murine o forms isa ofMMP-12 ta indicatethattheycleavedhuman tide substrates,althoughtheyareshorterand gnized bymurineMMP-12. We previously in substratebindingandthatdifferfrom one es. Ourresultsalsoshowthatthehemopexin seases, especially in COPD where MMP-12 is seases, especiallyinCOPDwhereMMP-12 e amino aciddifferencesontothe3Dstructure arenowrequired todetermine whetherthis explanationforwhysubstratesdesigned ogue duetosubtleamino aciddifferencesin fullyprotectedfrom tobacco smoke-induced ed more slowlythan full-length MMP-12 which wereinitiallydesignedforhuman murine MMP-12areunlikelytobothbe substrates developedforhuman neutrophil shown) lieintheso-calledS-loopand cation networksbetweendistalstructural K m andconsequentlyenhancethe MMP-12 inhuman pathogenesis 12s raise the question of the the cleavagesitesinprotein way ofregulatingthe α 1-PI and k cat / K m

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 cloned intothepET11a vector Taq/Pwo polymerase (Roche, Germany). Theresulting amplified fragments were digested and cloned into pCR4-TOPO (OpenBioSystems, USA)asatemplate for PCRamplification with corresponding toNde1andBamH1 restricti containing a6-Histag ATT GGATCC forward primer: ATC CATATG The catalyticdomain ofhuman (h-CAT MMP-12 Synthesis andpurificationof Full-length human MMP-1,-2 Materials Materials andmethods needed toconfirm its developing activity-basedprobes MMP-2 andMMP-9.Whileourresultssuggestth concentration ofMMP-12inbiologicalfluidsis for selectively measuring MMP-12 activity in The PP-16FRETsubstratebasedonthepep obtained frommouse model different proteinsubstrates showing thathuman andmurine MMP-12smay ha macrophages haveawiderspectrum ofMMPs(Sha have suggestedthatthepredominant macropha MMP-12 (Finlay activity inthelungsof peoplesuffering from remains controversialbecausesome studieshave other reagentswereofanalyticalgrade. human MMP-12wasboughtfrom were purchasedfrom R&DSystemsEurope(Lill Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New TCA GTG GTG GTG GTG GTG GTG TCC ATA CAG GGA CTG AAT G TCAGTGTCCATA CAGGGACTGAAT (5), 469-484. ,DOI :10.1515/hsz-2015-0254 et al. selectivity forMMP-12. , 1997),buttoincreasesinother Comment citer cedocument: sequence forfurtherpurification(E in vivo of COPD(Shapiro,2000). Substrate specificityofMMP-12 , -3,-7,-8,-9,-13,-14and-1 (MerckMillipore).The cDNAsequencewascheckedforthe Brought to youby|University ofCalifornia catalytic domainofMMP-12s GGG CCC GTATGGAGGAA, GGGCCC for visualizingMMP-12activity , emphasisestheneedforcauti Download Date |1/25/16 2:18 AM

Sino BiologicalInc(Interchim, Montluçon,France). All Authenticated 17 /33 on sites are underlined. We usedMMP-12cDNA tide sequenceRNALAVERT ge proteaseinmice isMMP-12,whilehuman much lowerthanthoseof tootherMMPs like emphysema isnotdueto demonstrated thattheincreased elastinolytic -MMP-12) wasamplified byPCRusing the e, France). Non-tagged catalytic domain of samples ofhuman originbecausethe at itmay provideavaluabletemplate for piro, 2003).This,togeth ve differentspecificityandconsequently MMPs andneutrophilelastase.Others 2 andfull-lengthmurine MMP-12 urofins, France). Thetriplets on ininterpreting the results in vivo, vivo, in and thereverseprimer:and AS may beuseful further studiesare er withourresults anincreasein Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 buffer, pH7.5containing Perray-en-Yvelines, France)exceptthatinclusio the cDNAcodingmouse MMP-12clonedinp The catalyticdomain ofmurineMMP-12wasexpr and theSensolyte was determined byactivesite sequencing theN-terminal amino acids.The western blottingusingapolyclonalanti- The identityofh-CAT-MMP-12wasconfir PD10 desaltingcolumn (GEHealthca mM). h-CAT-MMP-12wasthenrenaturedbybu same buffer.BoundHis-tagged (GE Healthcare,VélizyVillac affinity chromatography column connectedto imidazole at room temperature. Samples c buffer pH7.5containing150mM NaCl,5mMCaCl and dissolvedin50mMHEPE 8.0. Inclusionbodieswereisolatedbycentrifug collected bycentrifugation(9500 incubation withlysozyme (2mg/g ofpellet) and lysedbythreeheat-shockcycles(37° their OD High levelexpressionofh-CAT-MMP-12wasi the heat-shock method. Positiveclones weregrow MMP-12 plasmidintegrated into wasthen absence ofmutationsbynucleotidesequencin substrate. titration wasperformed usingthesame procedur buffer, pH8.0containing150mM NaCl,10mM CaCl hydrochloride and10mM Enzymeactivitywas imidazole. measured in50mM Tris-HCl Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New 600 reached0.6. Thecellswereharveste (5), 469-484. ,DOI :10.1515/hsz-2015-0254 ® 490MMPfluorogenicsubstrat Comment citer cedocument: 150 mM NaCl, 10mM CaCl Substrate specificityofMMP-12 titration usingtheMMPinhibi oublay, France)andthecolumn Brought to youby|University ofCalifornia S (N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid) MMP-12 waselutedwithagradientofimidazole (10-500 rpm, 4°C,30min) andsuspendedin20mM Tris-HCl,pH Download Date |1/25/16 2:18 AM

re, VélizyVillacoublay,France). Authenticated MMP-12 antibody(Abcam, Cambridge,UK),and E. coli 18 /33 ontaining MMP-12 werea C, 10min and-80°C, for 10min withgentleshaking.Thepelletwas molar concentration ofactiveh-CAT-MMP-12 g (Fasteris, Switzerland). ThepET11a/h-CAT- med bypolyacrylamide gelelectrophoresis, anÄKTAPurifierch e asthatusedforhuman MMP-12usingPJ-1 nduced byadditionofIPTGtoculturesonce n bodiesweredissolved ing this suspension at 9500 rpm for 30min ing thissuspensionat9500rpmfor ffer exchangeagainstHBNCbufferusinga BL21(DE3)competent cells(Novagen)by n inLBmedium andampicillinat37°C. e (Anaspec,Eurogentec). d bycentrifugation 4hoursafterinduction essed usingthesame protocolstartingfrom DONR vector(Gencopeia,Tebu-bio,Le 2 , 0.05%Brij35,7MGuHCl,10mM 2 2 and0.05%Brij35.Activesite , 0.05 % Brij 35,7Mguanidine , 0.05%Brij tor GM6001(MerckMillipore) washed extensively with the 10 min), followedby romatographic system pplied toaHis-Trap in 50mM Tris-HCl Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 using the Abz-peptidyl-EDDnpsubstrateweredeterm prepared inN,N-dimethylformamide anddiluted was checkedbystandardMALDI-TOFanalysis. St were alsosynthesized(>95%purity)byProteoGe linear gradient ofacetonitrile(0 E) andbyreverse-phasechromatography onaC18 desorption ionization-time of these substrates (Hirata et al. synthesizer (PSSM-8,Shimadzu Co.) (N-(9-fluorenyl)methoxycarbonyl) methodology Abz-peptidyl-EDDnp fluorogenicsubstrates by MMP-12. human andmouse MMP-12 (Devel because this sequenceis readily cleavedbymo We alsosynthesizedaseriesofsubstrates as fluorescentgroupandEDDnpN-(2,4-dini synthesize aseriesoffluorogenic deduced fromthecleavagesitesofsoluble literature (Table S1,Supplementary Inform the MEROPSdatabaseathttp: human MMP-12thatwerenotcl substrates to identifypeptidesequences that co all 20amino acidsintheP4toP4’regionof (P1, P2,etc.)ofsubstratesand The nomenclature ofSchechterandBerger(1967) Design andsynthesisof Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New , 1994).Thesynthesisstrategyresultedinglut ε 365 nm =17,300M-1cm-1 forEDDnp. et al. et (5), 469-484. ,DOI :10.1515/hsz-2015-0254 α 1-PI, human CXCL1andmouseCXCL5th , 2006).Lastly,weusedpeptidese Comment citer cedocument: et al. fluorogenic substrates Substrate specificityofMMP-12 , 1994).Substratepuritywascheck flight mass spectrometry (MALDI-TOF; Micromass, TofSpec- //merops.sanger.ac.uk/ (Rawlings enzyme subsites(S1,S2etc.).We analysedthefrequencies of Brought to youby|University ofCalifornia -60%) in0.075%trifluoroacetic eaved byotherMMPsThecleavage Abz-EDDnp substrateswithAbz( Download Date |1/25/16 2:18 AM

as previouslydescribed(Chagas Authenticated 19 /33 knownMMP-12cleavagesite derivedfrom thepeptidesequencePLGLEEA ation). TheconsensussequenceRPLALWRS trophenyl)ethylenediamine asquenchergroup. substrates wasusedasaleadsequenceto st MMPsand ispartiallyselectiveforhuman uld beusedtodesign ined bymeasuring theabsorbanceat365nm, were synthesizedbysolid-phaseFmoc nix SAS(Schiltigheim, France);theirpurity with activity buffer. The concentrations of isusedforindividualamino acidresidues ock solutionsofsubstrate(2-5mM)were column elutedat3ml/min witha25-min amine being the C-terminal residueinall using amultiple automated peptide quences spanningtheknownsitesin at arecleavedquiteefficiently acid. Some peptidesubstrates et al. ed by matrix-assisted laser ortho sites wereobtained from , 2014)and/orfrom the selective substrates for -aminobenzoic acid) et al. s invariousprotein , 1991;Hirata Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 cinnamic acidin33.3% acetonitrile,66.6%wate Daltonics). Samples weredilutedten-fo MALDI-TOF-MS spectrawereobtainedon an ElectroSpray Ionisation –IonTrap (ESI-IT) ma by Matrix Assisted LaserDesorptionIonisation 320 nm (Abzgroup)and 360nm(EDDnpgroup).Se TFA over25min. Elutedpeaksweremonito pump ataflowrateof3ml/m connected toanAgilent12seriesHPLC(hig were separatedbyreversephasechromatogra buffers. Reactionswerebloc Fluorogenic substrates(60µMfi Chromatographic procedures andid V Individual Software, LaJolla,CA,USA). experimental datatothefi V v = concentration of10nM.Undertheseconditions, conditions, usingasubstrateconc spectrofluorometer. Specificity constants ( measuring thefluorescenceat CaCl2, 0.05%Brij35.HydrolysisoftheAbz- Assays werecarriedoutat30°Cin50mM Enzyme assays andkinetic studies Menten rateequationusing of MMP-12wasalways10nM.Experimental data 12 weredetermined using8-10substrateconcentr k with [S] Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New cat m m /[E]t. = / K k obs m k ratio. Thek cat .S, where 0 and[S],beingthesubstrateconcentrations . [E]t,where[E]tisthefinalenzyme concentration,dividing K m and (5), 469-484. ,DOI :10.1515/hsz-2015-0254 k obs V m = obs values for thehydrolysisofAbz- values V forthefirst-order substrate hydrolysis wascalculatedby fitting Comment citer cedocument: m /K m ked byadding100µlaqueousTF . Integratingthisequationov Substrate specificityofMMP-12 rst-order lawusingGraphPad in, withalinear(0-60%,v/v)gr GraphPad Prism 5software. Brought to youby|University ofCalifornia nal) wereincubated withMMPs at30°Cintheirrespective λ ex Download Date |1/25/16 2:18 AM entration (5µM)farbelowthe

=320 nm and Authenticated entification of ld inasaturatedsolution of4-hydroxy- 20 /33 HEPES buffer, pH7.5, HEPESbuffer, phy onaC18column (2.1mm ×30mm, Merck) k h performance liquidchromatography) system red simultaneously at220nm (peptidebond), cat peptidyl-EDDnp substrateswasfollowedby the Michaelis-Mentenequationisreducedto: / ss spectrometry toidentify cleavage sites. K ations (0.2-20µM);thefinalconcentration UltraFlex Imass spectrometer (Bruker r and0.1%trifluoro m λ were fitted tothe hyperbolic Michaelis- attime 0andtime t,respectively.Since ) weredetermined underfirst-order em lected peptidefragments analysed were - Time ofFlight(MALDI-TOF)or =420 nm inaVarianCaryEclipse peptidyl-EDDnp substratesbyMMP- er time givesln[S]=- cleavagesites k adient ofacetonitrilein0.075% cat A (0.075%). Peptide fragments A (0.075%).Peptidefragments Prism 5software(GraphPad valueswereobtainedfrom K 150 mM 5mM NaCl, m andafinalenzyme k acetic acid.Matrix- obs by[E]tgavethe k obs .t +ln[S] α -cyano- 0

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 score, wereexamined with Pymol software(Schrödinger LLC, NewYork). 2011). Thetopfivemodels ofpeptideintera the receptor active siteto tool of the Rosettasuitethat allows full flexib FlexPepDock (http://flexpepdock.f catalytic domain ofMMP-12andtheundockedPP-16substratewassubmitted tothe stabilises the enzyme), whileth has thePhe171Aspmutation (f structure. Weusedthisprocedurebecause superimposing theVal-Glu-Argbackbonesof extended conformation usingPymol software with MMP-12S’subsites.Thepeptidesequenc bond (Bertini following cleavageofthecollagen-derived pep Gly peptidecomplex (2OXW PDB domain of MMP-12from the1JK3PDB(ProteinData Bank)file on theMMP-12 /Ile-Ala- PP-16)bysuperimposi RNALAVERTAS (substrate MMP-12 activesite.We first We useddockingstudiestohelpvisualizethein Docking studiesofpeptide substrates York, USA). Daltonics). Cleavagepeptides spectra wereprocessedandch ionization modeandatstandard/enhanced Daltonics) equippedwithanelectrosprayionsour spectra wererecordedonaHCTultraPTMDi spectra wereprocessedusingFlexAnalysis kit (Bruker)wasusedforca positive ionmode(1000lasershots) overthera method (Cadene sample solutionswerespottedontoagold-plat Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New et al. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 et al. , 2006).Thisrevealstheinteractions , 2000;Gabant Comment citer cedocument: be takenintoaccount during libration usingthenear-neighbor Substrate specificityofMMP-12 built amodelofMMP-12comple were assignedusingPawsve Brought to youby|University ofCalifornia arge deconvolutedusingDataAn at oftheJK3filedoesnot. requently introducedinto Download Date |1/25/16 2:18 AM urmanlab.cs.huji.ac.il) software

file). MMP-12forms anadduct et al. Authenticated 21 /33 the MMP-12structurefrom 3.3 softwarefrom BrukerDaltonics. ESI-IT-MS , 2008).Spectrawereacq resolution (8100 m/z persecond).ESI-IT-MS ility of bothpeptide residues andside-chains of ctions withMMP-12,sortedbytheirRosetta and positionedintheMMP-12activesiteby nge 500to 3500 m/z. Thepepmix Icalibration PP-16 onIle-Ala-Glyresiduesofthe2OXW scovery System massspectrometer (Bruker tide Pro-Gln-Gly-Ile-Al e ofsubstratePP-16wasconstructedinan ed sample probeusingtheultrathinlayer ce. Acquisitionswereca teraction of substrateswithresidues of the ng theX-raystructur the docking procedure (London between substrate residues P1'-P2'-P3' between substrateresiduesP1'-P2'-P3' rsion 8.5.0.3(ProteoMetrics,New recombinant MMP-12becauseit The complex formed betweenthe spotstrategy.MALDI-TOF-MS xed withthepeptidesequence alysis 3.1software(Bruker , ahigh-resolutiondocking with thispeptidemoiety uired inthereflectron the2OXWPDBfile a-Gly at the Gly-Ile Gly-Ile the at a-Gly rried outinpositive e ofthecatalytic et al. , Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Owen Parkes. Agreement LabexMab’Improve (ANR-10-LABX- Conseil RégionalCentre-ValdeLoireandth Régional Centre-ValdeLoire.RGwasawarde University ofRouen,France.ASLwassuppor acid sequenceanalyseswereperformed byth Protéomique» forMolecularBiophys oftheCenter Mass spectrometry analyseswereperformed byth Acknowledgements Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: Substrate specificityofMMP-12 Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

Authenticated 22 /33 e programme Investissement d’AvenirGrant e «Plateforme ProtéomiquePissaro»atthe ted byaPhDfellowshipfrom Conseil the d withapost-doctoral fellowship from the e «Plateforme deSpectrométrie de Masseet 53). TheEnglishtextwaseditedbyDr. ics inOrléans,France.N-terminal amino Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Deng, S.J.,Bickett,D.M.,Mitchell,J.L.,La Demedts, I.K.,Morel-Montero, A.,Lebecque,S.,Pacheco,Y.,Cataldo,D.,Joos,G.F., Bertini, I., Calderone, V.,Frag Banda, M.J.,Clark,E.andWerb, Z.(1980) Banda, M.J.,Clark,E.Sinha,S.,andTrav References Dean, R.A.,Cox,J.H.,Bellac,C.L.,Doucet, Curci, J.A.,Liao,S.,Huffman, M.D., Cobos-Correa, A.,Trojanek,J.B.,Diemer Chen, E.I.,Li,W., Godzik,A.,Howard, E.W. Chen, E.I.,Kridel,S.J.,Howard,W., Chagas, J.R.,Juliano,L.,andPrado,E.S. Cadene, M.,andChait,B.T.(2000).A Bhaskaran, R.,Palmier, M.O.,Lauer-Fields, Bertini, I.,Calderone,V.,Fragai,M.,Luch Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New macrophage interminating polym chemokines andgeneratesCCL2,-7,-8, Biol. Chem. specificity of human collagenase 3assessed 3rd, Neugebauer,J.,Pahel, G.,Weiner, M. induced sputum withCOPD.Thorax from patients Pauwels, R.A.,andBrusselle,G. hemopexin-like domains offull-lengthMMP-12.J.Am. Soc. Chem. and Svergun,D.I.(2008).Evidenceofreci inactivates human elastase withnativ 1314-1317. Macrophage-specific metalloelastase ( abdominal aortic aneurysms. J.Clin.Invest. el Expression andlocalizationofmacrophage Nat. Chem. Biol. Membrane-bound FRETprobevisualizesMM metalloproteinase-9. J.Biol.Chem. 4485-4491. unique substraterecognitionprofileformatr tetrapeptide substratesfor tissue a spectrometric analysis ofintegral V withexositesandhighactivity.J.Biol. Chem. (2008). MMP-12catalyticdomain recognizestrip Ed. Snapshots ofthereaction mechanism ofmatr subsite controls substrate selectivity of 45 , 7952-7955. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 275 , 31422-31427. 5 Comment citer cedocument: α , 628-630. e andoxidizedhuman 1-proteinase inhibitor.J.Exp.Med. Substrate specificityofMMP-12 Brought to youby|University ofCalifornia ai, M.,Jaiswal,R.,Luchinat, Download Date |1/25/16 2:18 AM

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66 ssion inamurine model of Gauthier, F.(2009).Structur identification of theirsubstrate specificities: relevance to , 970-976. ivity, lungmacrophage infiltra , 2599-2608. vant structural tory diseases.J.Biol.Chem. tory ratMMP-12. ProteinExpr.Purif. Authenticated 281 smoke-induced emphysema inmice. Science 24 /33 R. M.,andShapiro,S.D.(1997).Requirement D'Arcy, E.M., Masterson, , 11152-11160. Metallopeptidase-12/Macrophage elastase.In: K.,Walsh, M.C.,Huber, M., B.C.,Holman, rrano, H.,Wartelle, J.,Guyot,N.,Juliano,L., on, S.R.(2011).Matrix 1997). Hydrolysisofabroadspectrum of Soto-Quiros, M.E.,Avila, L.,Lasky-Su,J., ., Beau,F.,Cuniasse,P.,Yiotakis,A.,and ese, S.,Ihling,C.,Rusciani,A.,Jahreis,G., , C.E.(2010).Degradationoftropoelastin R. M.,andShapiro,S.D.(2006).Elastin lloproteinase expressionandproductionby peptides containingortho-aminobenzoyl-peptides L.,Kobayashi,D.K.,Kelley,G., ometry offull-lengthintegralmembrane g, J.,Kull,I.,Kere,Svartengren,M., specificities and release of matrikines. specificities andreleaseof erman, E.K.,Litonjua,A.A.,Weiss,S. e inhibitorsandsubstrateofmatrix ngs andG.S.Salvesen, (Eds.), Elsevier features. Methods D. L., Hersh, C.P.,Klanderman, B.J., D. L.,Hersh, enic proteasesubstrates:solid-phase function, andCOPDinhigh-risk n, D.(2001).Cloning,expression, emphysema. J.Clin.Invest. emphysema. al characterizationofmouse 1 , 299-308. 284 tion andprotectsagainst metalloproteinase-12 J.B.,FitzGerald,M. , 34084-34091. 46 156 21 , 54-61. , 268-274. , 240-247. 272 , 12189- 277 , Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Kridel, S.J.,Chen,E.,Kotra,L.P.,Howar Korkmaz, B.,Attucci,S.,Juliano,M.A., Neumann, U.,Kubota,H.,Frei Molet, S.,Belleguic, C.,Lena,H.,Germain, N.,Be Marchant, D.J.,Bellac,C.L.,Moraes,T.J. London, N.,Raveh,B.,Cohen,E.,Fathi,G Liu, M.,Sun,H.,Wang, X.,Koike,T.,Mishima, Lavigne, M.C.,Thakker,P.,Gunn,J.,Wong, A., Kridel, S.J.,Sawai,H.,Ratnikov,B.I.,Chen, Knight, C.G.,Willenbrock, F.,and Murphy, Kis-Toth, K.,Bacskai,I.,Gogolak,P.,Mazlo, Matsumoto, S.,Kobayashi,T.,Katoh,M.,Saito, Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New Nat. Protoc. surface ofhuman neutrophilswithfluorescen Gauthier, F.(2008).Measuring 266. Anal Biochem. specificity constantsfor collagenasesand Smith, J.W.(2002).Aunique for sensitivecontinuousassaysofthe Mca-Lys-Pro-Leu-Gly-Leu-Dp 36. lungs from patientswithchronicobstru M., Delaval,P.,andLagente,V.(2005).In 109-119. Nucleic AcidsRes. FlexPepDock webserver--high resolution modeling ofpeptide-protein interactions. metalloproteinase 12)withrheumato Fan, J.(2004).Associationofincreased 534-546. epithelial cellsexpress S. Q.,Pelker,J.W., Kobayashi,M.,and 23788-23793. matrix metalloproteinase from othermatr Substrate hydrolysisbymatrix me metalloproteinases inhibi Monocyte-derived dendritic Luo, Z.,Heilbron,K.,Norris,M.J.,Dua Bilawchuk, L.M.,Hendry,R.G.,Roberts aorta ofcholesterol-fedrabbits:relationshi Watanabe, T.(1998).Expressionandlocalizatio antiviral immunity.Nat. Med. Overall, C.M.(2014).Anewtranscriptiona Georgiadis, D.,Hegele,R.G.,Luo,H.,Granv (5), 469-484. ,DOI :10.1515/hsz-2015-0254 3 , 991-1000. 328 Comment citer cedocument: , 166-173. 39 , W249-253. Substrate specificityofMMP-12 and secreteMMP-12.Bioche ted byGM6001.Immunobiology Brought to youby|University ofCalifornia , K.,Ganu,V.,andLeppert,D.(2004).Characterization of Download Date |1/25/16 2:18 AM

20 a-Ala-Arg-NH2, afluorogenic cell subpopulationsusediffe substrate bindingmode disc elastase, proteinase3andcat , 493-502. talloproteinase-9. J.Biol.Chem. Authenticated id arthritis.ArthritisRheum. d, E.W.,Mobashery,S., 25 /33 matrix metalloproteinases. FEBSLett. Kalupov, T.,Jourdan,M.L.,Juliano,and ctive pulmonary disease. InflammRes. , Wadsworth, S.J.,Dufour,A., Butler,G.S., G. (1992).Anovelcoumarin-labelled peptide E. I.,Li,W., Godzik, ., andSchueler-Furman, O.(2011).Rosetta A., Szatmari, I.,andRajnavolgyi,E.(2013). A., Szatmari, p tolesiondevelopment. Am. J.Pathol. expression ofmacrophage ix metalloproteinases.J.Biol.Chem. Eppihimer, M.J.(2004).Human bronchial crease inmacrophage elastase(MMP-12)in H., Ikeda,K.,Watanabe, T.,Ochiai,N.,and on, A.G.,Cheung,C.T.,Ng,J.,Ang,L., n, W.,Bucyk,T.,Karpov,A.,Devel,L., S., Ikeda,Y.,Kobori,M.,Masuho,and tumor necrosisfactorconverting enzyme. Miyashiro, J.S.,Wasserman, A.M.,Wei, ille, D.J.,Dive,V.,McManus,B.M.,and rtrand, C.P.,Shapiro, l roleformatrix metalloproteinase-12 in ce resonanceenergytransfersubstrates. n ofmatrixmetalloproteinase-12 inthe m. Biophys.Res.Commun. 218 riminates membrane type-1 , 1361-1369. hepsin Gactivitiesatthe and Smith, J.W. (2001). substrate withincreased A., Strongin,A.Y.,and rent typesofmatrix 50 276 S.D.,Planquois,J. , 3112-3117. , 20572-20578. elastase(matrix 296 54 , 263- , 31- 324 277 153 , , , Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Zhou, H., Wu, Y., Jin, Y., Zhou, J., Zhang,C.,Ch Zhou, H.,Wu,Y.,Jin, Zhou,J., Wang, X.,Liang,J.,Koike,T.,Sun,H.,Ichikawa Wallace, A.M.,andSandford,J.( Taddese, S.,Weiss, A.S.,Jahreis,G.,Neubert Taddese, S.,Jung,M.C.,Ihling,Heinz,A., Taddese, S.,Jung,M.C.,Ihling,Heinz,A., Shipley, J.M.,Wesselschmidt, R.L.,Kobayashi, Shapiro, S.D.,Kobayashi,D.K.,andLey,T. Shapiro, S.D.(2003).Proteolysi Shapiro, S.D.(2000).Animal models forch Schirmer, H.,BassodaSilva,L., Schechter, I.,andBerger,A.(1967).Onthesize Rawlings, N.D.,Waller, M.,Ba Ratnikov, B. I.,Cieplak, P.,Gramatikoff, K Palmier, Doren,S.R.(2007). M.O.,andVan Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New obstructive pulmonary diseasesusceptib rabbits. Am. J.Pathol. metalloproteinase-12 enhances thedevelopment H., Watanabe, T.,Sasaguri,Y.,andFan,J. pulmonary disease?Am. J.Pharmacogenomics metalloproteinases: functionalimportance in domain 24.MatrixBiol. degradation ofhuman tropoela collagen type Iandtype III.Biochim. Biophys.Acta MMP-12 catalytic domain recognizes and cleavesatmultiple sitesinhuman skin collagen type Iandtype III.Biochim. Biophys.Acta MMP-12 catalytic domain recognizes and cleavesatmultiple sitesinhuman skin mice. Proc.Natl.Acad.Sci. USA Metalloelastase isrequiredformacrophage-med Chem. unique elastolyticmetalloproteinase produced klotho andmarlboro mice. Am. J. Biochem. Res.Commun. Biophys. of proteolytic enzymes, theirsubstrat Acad. Sci.USA Basis forsubstraterecognition M., Sun,Q.,Godzik,A.,Osterman, A.,Stec, fluorescence: overcoming theinner H. (2013).Geneticpolymorphi 1034. obstructive pulmonarydisease inaBr (2009). Matrixmetalloproteinase genepolymor 509. 268 , 23824-23829. (5), 469-484. ,DOI :10.1515/hsz-2015-0254 111 Comment citer cedocument: , E4148-4155. 165 Substrate specificityofMMP-12 28 Brought to youby|University ofCalifornia , 1375-1383. s inthelung.Eur.Respir.J. rrett, A.J.,andBateman, A. , 84-91. Teixeira, P.J.,Moreira,J.S Download Date |1/25/16 2:18 AM

and distinctionbymatrix meta stin byMMP-12andthegene sm ofmatrix metalloprotei Respir.Cell.Mol.Biol. 93 27 filter effect.Anal.Biochem. Authenticated , 3942-3946. , 157-162. 26 /33 es and inhibitors. NucleicAcids Res. ., Pierce, J.,Eroshkin, A.,Igarashi,., Pierce, Y.,Kazanov, ility: ameta-analysis. Sci.Rep. azilian population.Genet.Mol.Res. 2002). Geneticpolymorphisms ofmatrix , R.H.,andSchmelzer, Rapid determination ofenzyme kineticsfrom J.(1993).Cloning andcharacterizationofa ronic obstructivepulmonary disease:ageof Neubert, R.H.,andSchmelzer, C.E.(2010). Neubert, R.H.,andSchmelzer, C.E.(2009). D. K.,Ley,T.J.,a , T.,Kitajima, S.,Morimoto, M.,Shikama, (2004).Overexpressionof human matrix B.,Strongin,A., andSmith, J.W. (2014). e, L.,Jing,J.,Chen,Z., Li, W., Shen, and thedevelopmentof of theactivesiteinproteases.I.Papain. by human alveolarmacrophages. J.Biol. 2 phisms: lackofassociationwithchronic iated proteolysisandmatrix invasionin , 167-175. of inflammatory arthritis intransgenic 1804 1804 44 (2014).MEROPS:thedatabase ., Moreira,A.L.,andSimon, D. (Suppl.),30s-32s. , 731-739. , 731-739. 22 , 4-7. nase family andchronic lloproteinases. Proc.Natl. ration ofmatrikinesfrom nd Shapiro,S.D.(1996). 371 C. E.(2009).Invitro , 43-51. , 43-51. chronic obstructive 3 , 2818. 42 8 , D503- , 1028- Version postprint P2 P1' P1 P2 P3 P4 Group I Group II insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 regions spanningcleavagesitesbyMMP-12in of human MMP-12cleavagesitesasdetailedin Table 1 Tables andfigures Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New P2 b R L L E Dn EDDnp EDDnp A E E A E L G E EDDnp L L P EDDnp G R L EDDnp P Q Abz S Q PP-2 R EDDnp Abz W S Q R PP-2b L E W A E L L W A P Q EDDnp L L E S A P EDDnp E L E Abz P R L PP-21 EDDnp A Abz Q L S PP-19 Abz P Q E EDDnp R S PP-20 W R L Q W A Abz S L L R E P PJ-8 Q R L R S P L R R A W Abz L P L PP-22 Abz R A L PJ-5 P Abz R PJ-4 Abz PJ-1 Substrate P2 Az G A W S EDp EDDnp Q S R W L A L G R Abz PP-23 J3 b G L L R Q Dn EDDnp Q A R E L G L P G Abz PJ-3 name Peptide sequence Comments Abz-peptidyl-EDDnpfluorogenicsubstrat (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: Substrate specificityofMMP-12 Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

Authenticated 27 /33 P' 4 P' P5' P4' P3' ' naturalproteinsubstrates(GroupIV). Figure1(GroupsI,IIandIII)from es designedfromsequenceanalysis selectivity added atP4toincrease Derived from PP-2b. R al. by MMP-12 (Devel be cleavedefficiently previously reported to Peptide sequence by E at P4wassubstituted Derived from R PJ-1. R atP4 Derived from No PJ-1. at thosepositions AA negatively charged of evaluate theeffects to EE was replacedby sequence atP3'-P4' Derived from RS PJ-1. (Devel at thosepositions reported tobefavored because ithasbeen byEE P3' wasreplaced P2'- WR sequenceat Derived from PJ-1. E by replaced at P3'was Derived from R PJ-1. that position) E (same frequency at by replaced at P1was Derived from A PJ-1. that position) R (same frequency at by replaced at P2'was Derived fromW PJ-1. substrates sites insoluble cleavage analysis of derived from the Consensus sequence at thatposition P of evaluate theeffect P3to replaced Pat Derived from G PJ-1. substrates sites ofsoluble occurrence incleavage same frequency of P3' becauseithasthe at R was replacedby Derived from PP-2b. E , 2006) et al. et , 2006) et Version postprint EDDnp P I S M P I A E L F M A G Abz IV PP-17 Group EDDnp Q EDDnp A E E L E Q L A P E G R L Abz G L PJ-11 P G Abz PJ-10 Group III insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New EDDnp Q A A G EDDnp L G L P R Q A Abz G G PJ-7 L G L P R Abz PP-6

J9 b R L L G Q Dn EDDnp Q A G W L G L P R Abz PJ-9 (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: Substrate specificityofMMP-12 Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

Authenticated 28 /33 1987) 1-PI (Banda oxidized human alpha- mouse MMP-12 in by and (b) alpha-1-PI MMP-12 innative cleaved byhuman The FLpeptidebondis soluble susbtrates cleavage sitesof that positionin highest frequency at occuring withthe acid A, anamino by replaced at P3'was Derived from PP-6.G frequency wasretained with thehighest amino acidoccuring At eachposition,the substrates. and insoluble sites inbothsoluble cleavage analysis of the from inferrred Consensus sequence 1997) PI (Gronski 12 inhuman alpha-1- cleavage sitebyMPP- majorspanning the Peptide sequence substrate possible inthis group toPro wasnot because coupling Abz P4 was introducedat (highest frequency). G at P1 replaced G Derived from PP-2b. E substrate possible inthis group toPro wasnot because coupling Abz introduced atP4 that position). Gwas (highest frequency at atP2' replaced E Derived from PP-2b. R substrate possible inthis group toPro wasnot because coupling Abz at P4 G wasintroduced al. alpha-1 PI (Banda MMP-12 innative mouse cleaved by The PMbondis MMPs cleaved byother position insites that occurrence at has alowfrequency of W because MMP-12 selectivity towards W toincrease by replaced at P2'was Derived from PP-6.G , 1987) et al. et al. , , et Version postprint h-CAT-MMP-12 h-FL-MMP-12 m-CAT-MMP- mM optimise thecorresponding substrates. PJ-1 The leadpeptidesequences

Group IV Group III Group II Group I insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 MMP-12 (h-CAT-MMP-12), human full-length MMP-12 (h-FL-MMP-12), Specificity constantsweredetermined under fluorogenic substratesbyMMP12s. Table 2 Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New P1 Az R A A E T S EDp EDDnp EDDnp S A T EDDnp R E E T V A A A L I A V N Q R S A S Abz R P L A PP-16 A E E T Abz A V PP-15 S A Abz PP-14 PP-2b PP-15 PP-14 PP-16 PP-22 PP-20 PP-17 Abz-GAMF PP-17 Abz-GAMF PP-19 Abz-PL PP-21 Abz-E PP-23 Abz-RGLA PP-23 Abz-RGLA PJ-10 PJ-11 PP-2 PP-6 PJ-1 Abz-RPL PJ-4 Abz-RPL PJ-3 PJ-5 Abz-RPL PJ-8 PJ-9 Abz-RPL PJ-7

Specificity constants , PP-2b and PP-6 in groups I, II and III respectiv , PP-2bandPP-6ingroupsI,IIIII (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Abz-RNALA Abz-RNALA Abz-ASVATE Abz-ASVATE Abz-EAAPSSVIAATE-EDDnp Abz-EAAPSSVIAATE-EDDnp Abz-RPLG Abz-RPLG Abz-RPLG Abz-RPLG Abz-RPLA Abz-RPLA Abz-RPLA Abz-RPLA Abz-GPLG Abz-GPLG Abz-GPLG Abz-GPLG Abz-GPLE Abz-GPLE Abz-RPLA Abz-RPLA Abz-RPLG Abz-RPLG Abz-PLG Abz-PLG  PLA Comment citer cedocument: ↓    LEAI LEAI   A G A E A ↓ ↓ ↓ ↓ ↓ LW ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ LGGAQ-EDDnp LGGAQ-EDDnp LGAAQ-EDDnp LGAAQ-EDDnp LWEEQ-EDDnp LWEEQ-EDDnp ↓ LWESQ-EDDnp LWESQ-EDDnp ↓ LEEAQ-EDDnp LEEAQ-EDDnp LRQEDp 27 LWRSQ-EDDnp LERAQ-EDDnp LERAQ-EDDnp LREAQ-EDDnp LREAQ-EDDnp LEEA-EDDnp LEEA-EDDnp ↓ LEESQ-EDDnp VERTAS-EDDnp LGQEDp 55 LWGAQ-EDDnp LEEA-EDDnp LRQEDp 37 LWRSQ-EDDnp LRQEDp 69 LWRSQ-EDDnp LRQEDp 82 LWRSQ-EDDnp ↓ LRQEDp 71 LRRSQ-EDDnp

 LRAQ-EDDnp LRAQ-EDDnp Substrate specificityofMMP-12  P first-order conditions forthehydrolysis offluorogenic substratesby human cataly RQEDp 2 32 <2 RSQ-EDDnp Brought to youby|University ofCalifornia  MI-Dn 17 MSIP-EDDnp Download Date |1/25/16 2:18 AM

k cat / Authenticated K 29 /33 m for the hydrolysisofAbz-peptidyl-EDDnp forthe ely were modified atoneortwopositionsto ely were 3.6 9.3 24 74 23 74 10 64 11 61 murine catalytic domain ofMMP-12 (m-CAT-MMP-12) and <2 50 <2 170 <2 140                 

03 730 0.3 05 660 0.5 03 560 0.3 06 310 0.6 03 420 0.3 1 920 17 03 280 0.3 03 59 0.3 3 110 3 7 720 7 2 390 2 1 760 1 1 1600 1 2 700 2 3 190 3 1 110 1 2 620 2                      1 < <2 <2 10 1 < <2 <2 15 1 < 6.2 <2 14 5 2 <2 <2 5 8 8.4 80 1 < 8.5 <2 15 5 38 56 6 < 7.3 <2 62 2 < <2 <2 29 2 < 145 <2 29 3 < 3.9 <2 37 1 2.7 14 2 < 7.4 <2 20 2 < 4.7 <2 24 4 2 5.9 <2 4 5 2 2 <2 5 9 2 <2 <2 9 9 6 9 5 2 3.2 <2 5 1 13 19 4 2 7.4 <2 4 k cat

/ K -1 s m -1

 12    01 2.3 0.1  03 6.2 0.3 06 10 0.6 01 <2 0.1 1 <2 1

et al. murine (Dean CXCL5 12 cleavagesitein spanning humanMPP- Peptide sequence et al. murine (Dean CXCL5 12 cleavagesitein spanning humanMPP- Peptide sequence et al. human (Dean CXCL1 12 cleavagesitein spanning humanMPP- Peptide sequence tic domain of , 2008) , 2008) , 2008) m-FL-MMP-12 m-FL-MMP-12             0.1  0.3 0.1 0.6 0.4 0.4 0.1 0.3 0.1 0.2 7 1 3 Version postprint 110 117 4.7 0.55 10 32 9.1 0.29 Abz-RPLGLEEA-EDDnp Abz-PLGLEEA-EDDnp 10.3 64 720 19.7 0.19 18 PP-2 3.8 193 PP-2b PP-19 Abz-PLALWRSQ-EDDnp Abz-RPLALWEEQ-EDDnp 14.6 74 420 PP-20 12.3 20.2 0.17 12 PJ-8 Abz-RPLALEESQ-EDDnp 1.3 106 Abz-RPLALWESQ-EDDnp 74 560 6.8 Abz-RPLELWRSQ-EDDnp 760 PP-22 13.8 37 9.3 0.85 0.11 17 8.8 2.8 203 Abz-RPLALWRSQ-EDDnp 29.0 0.14PJ-5 15 1.4 159 42 14.4 82 920 27 15.6 PJ-1 0.33 23 5.6 359 0.26 24.9 9 61 1.5 190 60 7.5 Substrate Sequence 2.4 h-CAT-MMP-12 320 1600 h-FL-MMP12 µM µM mM insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 * and fluorogenic substratesbyhuman MMPs. Table 4 Individual parameters ( EDDnp fluorogenicsubstratesbyhuman MMP-12. Table 3  Minor cleavagesitesrepresentedlessthan 5 % of the primary cleav murine full-length MMP-12 (m-FL-MMP12). Menten equationusing Graph PadPr determined using were (h-FL-MMP-12) MMP-12 Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New standard deviation(S.D.)mi ofa MMP-12 MMP-2 MMP-7 MMP-9 MMP-13 MMP-13 MMP-9 MMP-7 MMP-2 MMP-12 PJ-4 PJ-1 PJ-5 mM PJ-8 PP-22 PP-20 PP-19 PP-21 k cat K / K m determinations were were <10%and m values as determined 2). conditionsvalues as (seeTable underfirst-order Specificity constants Kineticdata ( Abz-RPLALWRSQ-EDDnp Abz-RPLALWRSQ-EDDnp Abz-RPLALWEEQ-EDDnp Abz-RPLALWESQ-EDDnp Abz-RPLALWESQ-EDDnp Abz-EPLALWRSQ-EDDnp Abz-EPLALWRSQ-EDDnp Abz-RPLELWRSQ-EDDnp Abz-RPLELWRSQ-EDDnp Abz-RPLALRRSQ-EDDnp Abz-RPLALRRSQ-EDDnp Abz-RPLALEESQ-EDDnp Abz-RPLALEESQ-EDDnp Abz-PLALWRSQ-EDDnp Abz-PLALWRSQ-EDDnp K m (5), 469-484. ,DOI :10.1515/hsz-2015-0254 , k cat ) for thehydrolysis ofthecatalytic domain ofhumanMMP-12 (h-CAT-MMP-12) andfull-length human nimum oftwo distinctexperiments. Comment citer cedocument:

ism software. Results are shown as averages of of areshownasaverages Results software. ism K K m Substrate specificityofMMP-12

not indicatedfor thesakeofclarity. m ↓ Brought to youby|University ofCalifornia , and k k cat cat Download Date |1/25/16 2:18 AM increasing concentrationsof



and indicate major indicate secondaryminor and cleavagesitesby human MMP-12 respectively. k cat k cat k / / Authenticated -1 K K cat 1600 920 730 560 420 620 s 760 190 m 30 /33 -1 m

/ mM K age siteasassessed from HLPC forthehydrolysisofAbz-peptidyl-EDDnp         m 8 191 80 6 377 62 2 285 29 2 136 20 2 269 24 9 32 9 1 20 19 4 93 4 ) forthehydrolysisofselectedAbz-peptidyl- k cat

/ K -1 m s * -1 µM µM mM substrate (0.5-20 µM). Data substrate (0.5-20µM).Data at leasttwodistinctexperiments. Standard er         1 151 15 4 395 49 3 142 33 7 87 7 8 119 8 2 24 2 2 133 2 8 59 8 K m

k cat

 / K profiles. Resultsare shown a   -1      1 51 10 s 3 67 3 6 71 6 k 3 9 291 9 8 198 8 2 21 2 4 143 4 cat m -1

k cat were fittedtotheMichaeli < 2  /      -1

K  1 1200 10 s 1 2730 14 5 4420 54 1 140 1 1 336 1 1 47 1 2 1390 2 m -1

mM 989 k cat s an averages an / K rors for for rors        -1  m s 37 528 129 301 34 109 6 6 * -1

k cat s-

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 k residues P1andP1’.Data arevisualized usi sites in16distinctst unique cleavagesitesof44distinctsolublesu We determined thefrequency ofeachamino acidfrom positions P4toP4’withinthe74 MMP-12 Figure 1 SD of at least two distinct experiments. SD ofatleasttwo were determined underfirst-order conditions Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New cat PP-23 PP-17 PP-2 PP-2b PP-14 PJ-10 PJ-3 PP-16 PP-15 PP-6 PJ-11

PJ-9 PJ-7 / K m valuesfor thehydrolysis ofall 21substrates designedinth Frequenciesofamino acidsaroundth Abz-RGLALWRSQ-EDDnp Abz-RGLALWRSQ-EDDnp Abz-GAMFLEAIPMSIP-EDDnp Abz-GAMFLEAIPMSIP-EDDnp Abz-RPLGLEEA-EDDnp Abz-RPLGLEEA-EDDnp Abz-PLGLEEA-EDDnp Abz-ASVATELRAQ-EDDnp Abz-ASVATELRAQ-EDDnp Abz-GPLGLREAQ-EDDnp Abz-GPLGLREAQ-EDDnp Abz-GPLGLERAQ-EDDnp Abz-RNALAVERTAS-EDDnp Abz-RNALAVERTAS-EDDnp Abz-EAAPSSVIAATE-EDDnp Abz-RPLGLGGAQ-EDDnp Abz-GPLELEEAQ-EDDnp Abz-RPLGLWGAQ-EDDnp Abz-RPLGLWGAQ-EDDnp Abz-RPLGLGAAQ-EDDnp (5), 469-484. ,DOI :10.1515/hsz-2015-0254 ructural proteins(B).The scissi Comment citer cedocument: Substrate specificityofMMP-12 and compared withthoseobtained forfull-le Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

Authenticated 720 110 660 390 700 310 32 110 170 140 59 280 50 31 /33 is study by thefull-length form ofMMP-2,MMP-7,MMP-9 andMMP-1 ng aheatmap-like representation wherethe color              1 10 10 1 139 15 1 106 12 15 14 2 216 75 29 56 5 70 5 3 797 86 37 14 bstrates ofMMP-12(A)andthe176cleavage 4 33 <2 4 5 43 5 9 <2 <2 9 5 73 <2 5 e substratesitescleavedbyhuman le peptidebondiscleaved between           6 227 62 5 <2 5 1 2 8 <2 1 5 30 5 1 33 1 1 <2 1 7 56 7 6 80 6 5 184 5 8 8 8 ngth MMP-12(firstcolumn). Valuesare          2 270 2 1 15 1 7 62 7 1 14 1 1 1 160 15 8 5 133 5 3 750 3 <2 < 2 < 2 < 2 <2 < 2 < 2         3 135 3 3 2750 37 7 140 7 1 640 1 5 133 5 1 273 14 8 1620 8 5 905 5 252 62 87            10 14 11 16 8 121 25 61 means 91 1 6

 3

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 (red). Detailsofthisanalysisaregiven spectrum definestherelativefrequencyofeach using theprogram HBOND(http://c are red.(C)Hydrogenbondsbetweenpeptide (http://ligin.weizmann.ac.il/cma/). Contactareas <30Åareshowninbluewhilethose >30Å peptideandMMP-12 re RNALAVERTAS Arg isinlightorange.(B)Contactmap an are coloredorange,whileenzyme residuePhe171A 180A thatform ahydrophobicsandwich accommodatingthe peptidesubstrate P2Leuresidue (oxygen), blue(nitrogen),and sticks (top)orCPK(bottom). atoms of The standard orientation;them and methods). MMP-12 isshownwithitssolv (A) Thepeptide wasdocked in the active site Figure 2 Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New Dockingmodel ofRNALAVERTASpe (5), 469-484. ,DOI :10.1515/hsz-2015-0254 Comment citer cedocument: odeled peptidesubstrateRNALA Substrate specificityofMMP-12 light grey(hydrogen).Theen Brought to youby|University ofCalifornia Download Date |1/25/16 2:18 AM

ib.cf.ocha.ac.jp/bitool/HBOND/). in TableS1 (Supplementary Information). Authenticated 32 /33 ofMMP-12usingFlexPepDock(seematerials thepeptidearecoloredgreen(carbon),red amino acid from low(lightgrey) tohigh residues andMMP-12residues,calculated ent-accessible surface colored grey andin alysis of allinteractions between the sidues, ascalculatedwithCMA thatinteractsstronglywithsubstrateP5 ptide intheMMP-12 active site. zyme residuesHis172AandIle ↓ VERTAS isrepresented in

Version postprint insights intothesubstrate specificityof macrophage elastaseMMP-12. Biological Chemistry,397 1 hand24respectivelyin50mM instructions. Human andmouse pro-MMP-12were 12) by4-aminophenylmercuric acetate(APM (PRO-MMP-12)wasconvertedtothefulllengthformThe proformofMMP-12 (FL-MMP- Figure S1 0.05% (w/v)Brij35.Formouse MMP-12,5µMZnCl polyacrylamide gel(nonreducingconditions) ofMMP- Human andmouse (B)forms (A) the MaterialsandMethodssection. expressed in The His-tagged catalytic dom Lamort, A.-S.,Gravier, R.,Laffitte,A., Juliano,L.,Zani, M.-L.,Moreau, T.(2016). New SDS-PAGE analysisofthevarious E. coli (5), 469-484. ,DOI :10.1515/hsz-2015-0254 expressionsystemandpurifiedbyaffi Comment citer cedocument: Substrate specificityofMMP-12 Brought to youby|University ofCalifornia ain ofhuman ormouse Download Date |1/25/16 2:18 AM

Tris-HCl buffer,pH7.5,10mM CaCl Authenticated 33 / and coloured withCoomassie BlueG250. 12 (1µg)wereloadedontoa15%SDS forms ofhumanandmouse MMP-12. incubated with 1 mM APMA at 37 °C for incubatedwith1mM APMAat37°Cfor A) treatment followingthesupplier's 2 wasaddedintheactivationbuffer. nity chromatography asdetailed in MMP-12 (CAT-MMP-12)was 2 , 150 mM NaCl, , 150mM NaCl,