A True Auto Activating Enzyme

Supplemental Material can be found at: THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 39, pp. 33435–33444, September 30, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A True Autoactivating Enzyme STRUCTURAL INSIGHT INTO MANNOSE-BINDING LECTIN-ASSOCIATED SERINE PROTEASE-2 ACTIVATIONS *□ S Received for publication, June 2, 2005, and in revised form, July 13, 2005 Published,
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  ATrueAutoactivatingEnzyme STRUCTURALINSIGHTINTOMANNOSE-BINDINGLECTIN-ASSOCIATEDSERINE PROTEASE-2 ACTIVATIONS * □ S Received for publication,June 2, 2005, and in revised form, July 13, 2005 Published, JBC Papers in Press,July 21, 2005, DOI 10.1074/jbc.M506051200 Pe´terGa´l ‡1 ,VeronikaHarmat §1 ,AndreaKocsis ‡ ,Tu¨ndeBia´n ‡ ,La´szlo´ Barna ‡ ,Ge´zaAmbrus ‡ ,BarbaraVe´gh ‡ ,Ju´liaBalczer ‡ ,RobertB.Sim ¶ ,Ga´borNa´ray-Szabo´ § ,andPe´terZa´vodszky ‡2 Fromthe ‡ InstituteofEnzymology,BiologicalResearchCenter,HungarianAcademyofSciences,P.O.Box7,BudapestH-1518,Hungary,the § ProteinModelingGroup,HungarianAcademyofSciences,Eo¨tvo¨sLora´ndUniversity,Pa´zma´nyPe´terst.1A,BudapestH-1117,Hungary,and  ¶ MedicalResearchCouncilImmunochemistryUnit,DepartmentofBiochemistry,UniversityofOxford,Oxford0X13QU,UnitedKingdom Few reports have described in detail a true autoactivation proc-ess, where no extrinsic cleavage factors are required to initiate theautoactivation of a zymogen. Herein, we provide structural andmechanisticinsightintotheautoactivationofamultidomainserineprotease: mannose-binding lectin-associated serine protease-2(MASP-2), the first enzymatic component in the lectin pathway of complementactivation.WecharacterizedtheproenzymeformofaMASP-2 catalytic fragment encompassing its C-terminal threedomainsandsolveditscrystalstructureat2.4Åresolution.Surpris-ingly, zymogen MASP-2 is capable of cleaving its natural substrateC4, with an efficiency about 10% that of active MASP-2. Compari-sonofthezymogenandactivestructuresofMASP-2revealsthat,inadditiontotheactivationdomain,otherloopsoftheserineproteasedomain undergo significant conformational changes. This addi-tional flexibility could play a key role in the transition of zymogenMASP-2 into a proteolytically active form. Based on the three-di-mensional structures of proenzyme and active MASP-2 catalyticfragments, we present model for the active zymogen MASP-2 com-plex and propose a mechanism for the autoactivation process. Extrinsic activating factor-initiated autoactivation of a zymogen is aclassic textbook case. To date, however, few reports have described atrue autoactivation process, where no extrinsic cleavage factors arerequired and the autoactivating capacity is an inherent property of thezymogen.Aphysiologicallyimportantexampleoftrueautoactivationisthe initiation of the complement cascade activation.The complement system is one of the proteolytic cascade systemsfound in the blood plasma of vertebrates. It provides the first line of immune defense against invading pathogens. The complement systemis a sophisticated network of proteins (involving more than 30 compo-nents), which can be activated via three different routes: the classical,the lectin, and the alternative pathways (1). Activation of the comple-ment system culminates in the destruction and clearance of invadingmicroorganismsanddamagedoralteredhostcells.Thecentralcompo-nentsofthesystemaremultidomainserineproteases,whicharepresentin zymogen forms and activate each other in a cascade-like manner (2).In the case of the classical and lectin pathways, a recognition mole-cule binds to a specific target, and this provides the activation signalthat is transmitted to serine protease zymogens, which in turn initiatethe cascade (3).Mannose-bindinglectin(MBL) 3 istherecognitionsubunitofthelec-tin pathway (4). MBL binds to carbohydrate arrays (mainly to mannoseand N  -acetylglucosamine residues) on the surface of pathogens, whichresults in the autoactivation of MBL-associated serine protease-2(MASP-2) (5, 6). Activated MASP-2 then cleaves C4 and C2, the pre-cursors of the C3 convertase enzyme complex. MASP-2 is the onlyknown MBL-associated protease that can directly initiate the comple-ment cascade, playing a key enzymatic role in the lectin pathway.The MBL-associated serine proteases together with C1r and C1s, theproteasesubcomponentsofthefirstcomponentoftheclassicalpathway(C1), form a family of enzymes with identical domain organization (7).The C-terminal trypsin-like serine protease (SP) domain is preceded byfive noncatalytic modules. At the N terminus, there is a C1r/C1s/seaurchin Uegf/bone morphogenic protein (CUB) domain followed by anepidermalgrowthfactor(EGF)-likemoduleandasecondCUB domain.This N-terminal CUB1-EGF-CUB2 region is responsible for the inter-subunitinteractions( e.g. interactionbetweentheproteasesandtherec-ognition subunits). The following two complement control proteinmodules (CCPs), which associate directly with the SP domain, stabilizethe structure of the SP domain and are involved in the proteolyticprocess.In the case of C1s and MASP-2, which share almost the same sub-strate specificity, the CCPs were shown to provide accessory bindingsites for the C4 substrate and thereby increase catalytic efficiency (8, 9).The CCPs, however, do not increase the efficiency of C2 cleavage, indi-cating that the two substrates bind to different regions of the enzymes.MASP-1, MASP-2, and C1r are capable of autoactivation, where thezymogenproteasesbecomecleavedandactivatedwithoutthecontribu-tion of any extrinsic cleavage factor. The autoactivation is an inherentproperty of the serine protease domains, the other modules are notinvolved in this process (9, 10). Our main priority was to characterizethe structural background of the autoactivation process.Recently, the three-dimensional structure of the activated MASP-2CCP2-SP fragment has been solved (11). The structure revealed the *  This work was supported by Hungarian National Science Foundation (OTKA) Grants T046444 andTS044730, the Hungarian Ministry of Health (EU¨Tana´cs555/2003), andNatImmuneA/SDenmark.Thecostsofpublicationofthisarticleweredefrayedinpartby the payment of page charges. This article must therefore be hereby marked“ advertisement  ”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. Theatomiccoordinatesandstructurefactors(code1ZJK)havebeendepositedintheProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics,RutgersUniversity,New Brunswick,NJ( □ S  The on-line version of this article (available at contains supple-mental Fig. 1 and TABLE ONE. 1  These two authors contributed equally to this work. 2  Towhomcorrespondenceshouldbeaddressed:Pe´terZa´vodszkyInst.ofEnzymology,Biological Research Center, Hungarian Academy of Sciences, P.O. Box 7, BudapestH-1518, Hungary. Tel.: 36-1-209-3535; Fax: 36-1-466-5465; E-mail: 3  The abbreviations used are: MBL, mannose-binding lectin; MASP-2, MBL-associatedserine protease-2; SP, serine protease; CUB, C1r/C1s/sea urchin Uegf/bone morpho-genic protein domain; EGF, epidermal growth factor; CCP, complement control pro-tein module.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 39, pp. 33435–33444, September 30, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. SEPTEMBER 30, 2005ã VOLUME 280ãNUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY  33435   b  y  g u e s  t   , onA  pr i  l  1  9  ,2  0 1 1 www. j   b  c . or  gD  ownl   o a d  e d f  r  om Material can be found at:  background of some important physiological properties of the MASP-2enzyme. In this report, we characterize the zymogen catalytic fragment(CCP1-CCP2-SP) of MASP-2 and describe its x-ray structure. Basedupon the zymogen and active structures, we present models for theautoactivating complex and propose a mechanism for autoactivation. MATERIALSANDMETHODS  Mutagenesis, Expression, and Purification of MASP-2 CCP1-CCP2-SP R444Q Mutant  —Mutagenesis was performed with theQuikChange  site-directed mutagenesis kit (Stratagene, La Jolla, CA)according to the manufacturer’s instructions. Recombinant plasmid forexpression of wild type MASP-2 CCP1-CCP2-SP was used as template.Recombinant protein expression and renaturation were performed asdescribed earlier (9). The renatured protein solution was concentratedonultrafiltrationmembrane(MilliporeCorp.,Bedford,MA),anditwascarried through its isoelectric point (pI 5.6) by dropping it into a 0.5 M sodiumacetatebuffer(pH5.0).Thesolutionwasdialyzedagainst50m M NaOAc,0.5m M EDTA(pH5.0)andfilteredona0.45-  mnitrocellulosemembrane. The renaturated protein was purified on a Mono S HR 5/5column (Amersham Biosciences). It was eluted with a linear NaCl gra-dientfrom200to600m M .Thecollectedfractionwasonceagaincarriedthroughtheisoelectricpointbydroppingitintoa1 M HEPESbuffer(pH7.4), and it was dialyzed against 20 m M HEPES, 145 m M NaCl, 5 m M EDTA (pH 7.4). The purification steps were monitored on SDS-PAGE.  Purification of C4 —Human C4 was prepared from 20 ml of freshserum according to the methods of Dodds (12). The obtained proteinwas  70% pure, and it was dialyzed against 20 m M HEPES, 145 m M NaCl, 0.5 m M EDTA (pH 7.4). Small C4 aliquots were frozen in liquidnitrogen and kept at  80 °C. They were thawed only once and used upwithin 3 days.  Purification of Wild Type Zymogen MASP-2 CCP1-CCP2-SP  —Theexpression, solubilization, renaturation, and dialysis were performedaccordingtoRef.9.Theproteinwaspurifiedatthesameconditionsasitwas in the case of the R444Q mutant except that the entire procedurewas carried out at 4 °C. Zymogen aliquots (1.93  M ) were frozen inliquid nitrogen and kept at  20 °C. Right before usage, zymogen ali-quots, thawed and kept on ice and were consumed within 24 h.  Autoactivation of Wild Type Zymogen MASP-2 CCP1-CCP2-SP  —Autoactivation experiments were carried out under physiological conditions.TheconcentrationofzymogenMASP-2CCP1-CCP2-SPwas1.93  M .12–14samplesweretakenatvarioustimepointswithin60minfromthebeginningof incubation.Anestimatedhalf-lifewasgivenforthezymogenbymeasuringthediminutionoftheCCP1-CCP2-SPchainandtheappearanceoftheSPdomainonreducingSDS-PAGE.ThequantificationofthesedatawasmadebyusingaGELDOC1000instrumentandMolecularAnalystsoftwarefordensitometriccalculations (Bio-Rad). 2–4 parallel experiments were analyzed to determinethehalf-lifeofwildtypezymogen.  Activation of MASP-2 CCP1-CCP2-SP R444Q Mutant  —The R444Q(2.95  M ) mutant was activated by thermolysin as described in Ref. 13. C4 Cleavage —To measure the kinetic parameters of the C4 cleavageby MASP-2 CCP1-CCP2-SP R444Q mutant and by the thermolysin-activated mutant, they were incubated with C4 at 37 °C. Serial dilutionswere made from mutant and substrate to find the optimal, well charac-terizableconditions.Theconcentrationwas2.85  10  8 M ,6.11  10  9 M , and 6.08  10  7 M for the uncleaved R444Q mutant, the thermoly-sin-activated R444Q mutant, and the C4 substrate, respectively. Typi-cally,11–13samplesweretakenwithin55minfromthebeginningofthereaction at various times. Data from 2–4 independent measurementswereusedforthecalculations.Thekineticparametersweredeterminedby visualizing and measuring the diminution of the  chain of C4 onCoomassie-stained SDS-PAGE using a GEL DOC 1000 instrument andMolecular analyst software (Bio-Rad) for densitometric calculations.The reactions were assumed to be of the Michaelis-Menten type. Thekineticconstants k  cat ,  K  m ,and k  cat /  K  m wereestimatedbyunbiased,non-linear regression methods, regressing the data on the following equa-tion: t   ([S 0 ]  [S]   K  m  ln([S 0 ]/[S]))/( k  cat  [E 0 ]).Inthepresenceof C1inhibitor,nocleavagecouldbedetected,sothisreactionwasusedasa negative control. The Effect of R444Q Mutant on the Autoactivation of Zymogen MASP-2 CCP1-CCP2-SP  —Wild type zymogen MASP-2 CCP1-CCP2-SP (0.064  M ) was incubated with a 10-fold excess of R444Q(0.64  M ) mutant. Serial dilutions of the wild type zymogen were madeto find a concentration at which the rate of autoactivation was slowenough. The incubations were carried out in 20 m M HEPES, 145 m M NaCl, 0.5 m M EDTA (pH 7.4) buffer at 37 °C for 3 h. All of the sampleswere visualized on SDS-PAGE and were analyzed by densitometry asdescribed above. The appearance of the SP domain was followed. Cleavage of Synthetic Substrate —The cleavage rates of MASP-2CCP1-CCP2-SP fragments on the synthetic substrate benzyloxycar-bonyl-Gly-Arg-S-benzyl (MP Biomedicals Inc., Aurora, OH) wereobtained as described in Ref. 11.  Differential Scanning Calorimetry —Calorimetric measurementswereperformedonaVP-DSC(MicroCal)differentialscanningcalorim-eter. Denaturation curves were recorded between 20 and 80 °C at apressure of 2,5 atm, using a scanning rate of 1 °C/min. The proteinconcentration was set to 0.2 mg/ml. Samples were dialyzed against 20m M Hepes (pH 7.4), 145 m M NaCl, and the dialysis buffer was used as areference. Heat capacities were calculated as outlined by Privalov (14). CrystallographicstudiesonzymogenMASP-2R444QMutant  —Crys-tals of the zymogen MASP-2 CCP1-CCP2-SP R444Q mutant fragmentwere grown using the hanging drop method at 20 °C. Crystals wereobtained by mixing 2  l of reservoir solution and 2  l of protein solu-tion.Thereservoirsolutioncontained20%polyethyleneglycol6000,0.2 M NaCl, 10% glycerol, and 0.1 M Tris-HCl, pH 8.0. The protein solutioncontained1mg/mlMASP-2fragmentinabufferof20m M Tris/HCl,pH7.4, and 0.03% NaN 3 .Data were collected using the ID14 EH4 beam line at the EuropeanSynchrotron Radiation Facility at cryogenic temperatures. Data wereprocessed with the XDS program package; they were scaled, merged,and reduced with XSCALE (15).The structure was solved by molecular replacement using the pro-gram MOLREP (16) of Collaborative Computing Project 4 (17). The SPdomain of the MASP-2 activated structure (11) (Protein Data Bankaccession code 1Q3X) was used as a search model. Refinement wascarried out with the REFMAC5 program (18), using restrained maxi-mum likelihood refinement and TLS refinement (19). ARP (20) wasused for automatic solvent building. Model building was carried outusing the O program (21). The final model contains protein residues296–686 with the exception of residue 661. The stereochemistry of thestructure was assessed with PROCHECK (22). Data collection andrefinement statistics are shown in TABLE ONE.  Molecular Modeling of the Enzyme-Substrate Complex of MASP-2 —WedockedtheP4-P3  (Thr 441 –Gly 447 )segmentofzymogenMASP-2tothesub-stratebindingsiteoftheactivestructure.TheAutoDock3.05(23)programhasbeen used for docking the zymogen MASP-2 heptapeptide fragment to theactive form. The charges were assigned to the structures by using SYBYL 6.5(Tripos Associates Inc., St. Louis, MO). The initial conformation of the frag-ment was built and optimized by SYBYL 6.5. AutoDockTools was used todefinetherotablebondsofthefragment.WeusedaLamarckiangeneticalgo-rithm for the docking with local search, parameterized according to previoussystematic optimization studies (24) (ga_run  200; generation  27.000;ga_num_evals  25.000.000;ga_pop_size  100,numberofactivetorsion   AutoactivationofMASP-2 33436 JOURNAL OF BIOLOGICAL CHEMISTRY  VOLUME 280ãNUMBER 39ã SEPTEMBER 30, 2005   b  y  g u e s  t   , onA  pr i  l  1  9  ,2  0 1 1 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   18).InordertofitthezymogenMASP-2tothedockedfragment,wemutatedbackthestructure insilico accordingtothewildtype(Q444R).Tohaveareal-istic model, we built a conformational library of the flexible loop of zymogenMASP-2 that contains our heptapeptide fragment (Arg 439 –Gly 448 ) by SwissPDBviewer(25).Aselectionofenergeticallyfavorableloopconformationshasbeen root mean square deviation-fitted to the docked heptapeptide. TheGly 442 –Tyr 446 peptidefragmentofzymogenMASP-2wasreplacedbythecor-respondingdockedfragment.Theobtainedcomplexstructureofzymogenandactivated MASP-2 was energy-minimized, relaxed at 310 K by moleculardynamics simulation in water using GROMACS (26). Coordinates of theMASP-2-MASP-2complexmodelareavailableuponrequest. RESULTSANDDISCUSSION CharacterizationoftheRecombinantZymogenCCP1-CCP2-SP FragmentofMASP-2  DesignofStableZymogenMASP-2CCP1-CCP2-SP  —The purpose of the present study was to investigate the autoactivation mechanism of the MASP-2 zymogen at various structural levels. MASP-2 possessesinherent autoactivating capacity and requires no extrinsic enzymaticfactors for the autoactivation to occur. Wild type MASP-2, similarly toothertrypsin-likeproteases,canbeactivatedthroughthecleavageofanArg-Ile bond at the N-terminal region of the catalytic SP domain. Wedesigned, constructed, and expressed an R444Q mutant of the catalyticfragment of MASP-2, thereby ensuring that the protein does notundergo autoactivation during the enzymatic characterization, crystal-lization, and structure determination processes. Gln was chosen on thebasisofisomorphicreplacement(27)andwaspredictedtohavetheleastprobability to interfere with the protein fold. Stability of Zymogen MASP-2 CCP1-CCP2-SP R444Q Mutant  —Weexpressed the R444Q mutant of the catalytic CCP1-CCP2-SP fragmentofMASP-2in  E. coli cells.Thewildtypeactivatedformofthisfragmentof MASP-2 has already been successfully expressed in the same expres-sionsystem,anditsbiochemicalpropertieshavebeencharacterized(9).After renaturation, the R444Q mutant was purified to homogeneity byionexchangechromatography.Thepurifiedproteinmigratedasasingleband of 44 kDa on the reducing SDS-PAGE (Fig. 1), indicating that nocleavage occurred within the polypeptide chain during expression andpurification. In contrast to the R444Q mutant, the wild type fragmentwasfullyactivatedafterthesametreatment.Toassessthestabilityofthepurified R444Q mutant, it was labeled with 125 I and was incubated witheither buffer or human plasma at 37 °C for 24 h. The samples were runonSDS-PAGE,andtheproteinswerevisualizedbyautoradiography.Nocleavageproductcouldbeobserved(datanotshown)demonstratingthestability of the R444Q zymogen even upon prolonged incubation atphysiological conditions.  Folding of Zymogen MASP-2 CCP1-CCP2-SP R444Q Mutant  —Tofurther characterize the folding and stability of our stable zymogenR444Q mutant, we measured the melting profile of the active wild typeand the zymogen R444Q mutant by differential scanning calorimetry.As the melting curves demonstrate (Fig. 2), both fragments show asharp, cooperative melting transition, indicating a compact, foldedstructure. The melting point of the active fragment (50.8 °C) is 2.6 °Chigher than that of the zymogen form (48.2 °C), and the calorimetricenthalpy change is also larger in the case of the active species. Thisdifference can be explained by the stabilization effect of the activationprocess. During activation, the loosely bound, flexible loops of the acti- vation domain become part of the more compact activated structure.ThezymogenmutantMASP-2fragmentshowednodetectableactiv-ity on synthetic substrate (benzyloxycarbonyl-Gly-Arg-S-benzyl) evenat very high levels of enzyme concentration (  200  M ), indicating thatthe catalytic machinery is disrupted in the zymogen and there is noactive trypsin-like serine protease contamination in the purifiedmaterial.To confirm that thezymogen mutant MASP-2 CCP1-CCP2-SP frag-ment is correctly folded and can be converted into an active enzyme, itwas treated with thermolysin (a non-trypsin-like) protease to specifi-cally cleave the Gln 444 -Ile 445 bond (13). The R444Q mutant was acti- vated using limited proteolysis by thermolysin (Fig. 1), giving rise to anactive MASP-2 species with activity on a synthetic and a protein sub- TABLEONE DatacollectionandrefinementstatisticsofthezymogenR444QmutantformofMASP-2CCP1-CCP2-SPfragment Crystal parameters Space group P2 1 2 1 2 1 Cell constants a  47.665 Å, b  72.689 Å, c  110.989 ÅData quality Resolution range (last resolution shell) 28.868–2.18Å (2.25–2.18 Å)  R meas a 0.099 (0.575)Completeness 88.1% (43.1%)No. of observed/unique 151,887/18,330 (1508/800)  I  /   (  I  ) 15.13 (1.90)Refinement residuals  R 0.207  R free b 0.253Model quality Root mean square bond lengths (Å) 0.005Root mean square bond angles(degrees)0.875Root mean square general planes (Å) 0.002Ramachandran plot: residues in core/allowed/disallowed regions272/51/0Model contentsProtein residues 390Protein atoms/water molecules 2910/84Residues in dual conformations 1Residues with disordered side chains 23Disordered residues 1 a  R meas  (  h ( n /( n  1)) 0.5   j    I  h   I  hj   )/(  hj   I  hj  ) with   I  h  (   j   I  hj  )/ n  j  . b 5.1% of the reflections in a test set for monitoring the refinement process. FIGURE1. Coomassie-stainedSDS-PAGEofwildtypeandR444QmutantMASP-2CCP1-CCP2-SPfragment. Lane1 ,molecularmassmarkers; lane2 ,wildtypeactivatedMASP-2CCP1-CCP2-SP; lane3 ,zymogen MASP-2 CCP1-CCP2-SP R444Q mutant; lane 4 , thermolysin-activated MASP-2 CCP1-CCP2-SP R444Q mutant.  AutoactivationofMASP-2 SEPTEMBER 30, 2005ã VOLUME 280ãNUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY  33437   b  y  g u e s  t   , onA  pr i  l  1  9  ,2  0 1 1 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   stratecomparablewiththatofthewildtypeMASP-2fragment(TABLETWO).TherecoveryofenzymaticactivityoftheR444Qmutantfollow-ing thermolysin cleavage underlined that it is suitable for studying thestructural and functional properties of zymogen MASP-2. The Cleavage of C4 by Zymogen MASP-2 CCP1-CCP2-SP R444Q Mutant  —Previous studies demonstrated that a zymogen MASP-2(S633A) mutant was able to form a complex with C4, a natural proteinsubstrate of wild-type MASP-2 (29). This is most probably due to theaccessory C4 binding sites on the CCP2 module of MASP-2 (9). WeincubatedourstablezymogenR444QMASP-2withhumanC4at37 °C.To our surprise, zymogen MASP-2 was able to cleave C4 with a highefficiency (Fig. 3, TABLE THREE). This activity was completely abol-ished in the presence of C1 inhibitor, indicating that it was mediated byzymogen MASP-2 and not by other (potentially contaminating) prote-ase. The fact that the K  m  values for the zymogen and the active enzymeare in the same range indicates that the accessory C4 binding site ispresent on both forms of the enzyme, and it is not affected by theconformation change of MASP-2 activation. Since zymogen MASP-2showed no activity on synthetic substrate but was shown to cleave C4,we argue that the one-chain zymogen form of MASP-2 can adopt anactive-like conformation, and this conformational change may beinduced by the large protein substrate C4. Previously, it was shown thattrypsinogen can be converted into an active state upon strong ligandbinding ( e.g. pancreatic trypsin inhibitor and Ile-Val dipeptide) withoutproteolyticcleavage(30).Aphysiologicallyimportantexampleofapro-teolytically active serine protease zymogen is tissue-type plasminogenactivator, which has significant (10–20%) activity relative to the two-chain form (31). The proteolytic activity of zymogen MASP-2 could beresponsible for the first step of the autoactivation process, where azymogen MASP-2 molecule cleaves and activates another zymogenMASP-2 molecule.  Autoactivation of MASP-2 —The observed rate of MASP-2 autoacti- vation is concentration-dependent. At low concentrations (  0.1  M )during renaturation, the catalytic fragment of wild type MASP-2remains zymogen for several days. However, rapid autoactivationoccurs during the subsequent concentration and purification steps (9).To prepare wild type zymogen MASP-2, we adjusted the pH to 5.0immediately after renaturation and performed the subsequent chro-matographic steps at 4 °C. At pH 5.0, the histidine residue in the cata-lytictriadbecomesprotonated,andtherateoftheproteolysisdecreases TABLETWO CleavageratesofMASP-2CCP1-CCP2-SPanditsR444Qmutantonbenzyloxycarbonyl-Gly-Arg-S-benzyl(Z-Gly-Arg-S-Bzl)syntheticsubstrateandonC4proteinsubstrate Enzyme Z-Gly-Arg-S-Bzl k  cat /  K  m C4 k  cat /  K  m  M   1  s  1 MASP-2 CCP1-CCP2-SP 9.40  10 5  6.2  10 4 5.50  10 5  5  10 4 a MASP-2 CCP1-CCP2-SP R444Q activated by thermolysin 1.30  10 5  1.7  10 3 8.50  10 5  2.5  10 5 MASP-2 CCP1-CCP2-SP R444Q — b 7.36  10 4  1.0  10 4 a Data from Ambrus et al. (9). b Below the detection limit of the assay. FIGURE2. DSCmeltingprofilesofwildtypeandR444Q mutant MASP-2 CCP1-CCP2-SP frag-ment. Shown is excess transition heat capacity of the wild type ( solid line ) and R444Q mutant( dashed line )fragments.Meltingtemperaturesareindicated.FIGURE 3. Cleavage of C4 by MASP-2 CCP1-CCP2-SP R444Q mutant. Incubation times areindicatedinminutes.The  and  chainsofC4andthe digestion product,   , are indicated by the arrows .  AutoactivationofMASP-2 33438 JOURNAL OF BIOLOGICAL CHEMISTRY  VOLUME 280ãNUMBER 39ã SEPTEMBER 30, 2005   b  y  g u e s  t   , onA  pr i  l  1  9  ,2  0 1 1 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 
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