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• W2097771036 abstract "Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 Å resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process. Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 Å resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process. Extrinsic activating factor-initiated autoactivation of a zymogen is a classic textbook case. To date, however, few reports have described a true autoactivation process, where no extrinsic cleavage factors are required and the autoactivating capacity is an inherent property of the zymogen. A physiologically important example of true autoactivation is the initiation of the complement cascade activation. The complement system is one of the proteolytic cascade systems found in the blood plasma of vertebrates. It provides the first line of immune defense against invading pathogens. The complement system is a sophisticated network of proteins (involving more than 30 components), which can be activated via three different routes: the classical, the lectin, and the alternative pathways (1Morley B.J. Walport M.J. Complement Factsbook. Academic Press, London1999Google Scholar). Activation of the complement system culminates in the destruction and clearance of invading microorganisms and damaged or altered host cells. The central components of the system are multidomain serine proteases, which are present in zymogen forms and activate each other in a cascade-like manner (2Sim R.B. Tsiftsoglou S.A. Biochem. Soc. Trans. 2004; 32: 21-27Crossref PubMed Scopus (160) Google Scholar). In the case of the classical and lectin pathways, a recognition molecule binds to a specific target, and this provides the activation signal that is transmitted to serine protease zymogens, which in turn initiate the cascade (3Gál P. Ambrus G. Curr. Protein Pept. Sci. 2001; 2: 43-59Crossref PubMed Scopus (26) Google Scholar). Mannose-binding lectin (MBL) 3The abbreviations used are: MBL, mannose-binding lectin; MASP-2, MBL-associated serine protease-2; SP, serine protease; CUB, C1r/C1s/sea urchin Uegf/bone morphogenic protein domain; EGF, epidermal growth factor; CCP, complement control protein module. is the recognition subunit of the lectin pathway (4Holmskov U. Thiel S. Jensenius J.C. Annu. Rev. Immunol. 2003; 21: 547-578Crossref PubMed Scopus (655) Google Scholar). MBL binds to carbohydrate arrays (mainly to mannose and N-acetylglucosamine residues) on the surface of pathogens, which results in the autoactivation of MBL-associated serine protease-2 (MASP-2) (5Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B.M. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (752) Google Scholar, 6Schwaeble W. Dahl M.R. Thiel S. Stover C. Jensenius J.C. Immunobiology. 2002; 205: 455-466Crossref PubMed Scopus (123) Google Scholar). Activated MASP-2 then cleaves C4 and C2, the precursors of the C3 convertase enzyme complex. MASP-2 is the only known MBL-associated protease that can directly initiate the complement cascade, playing a key enzymatic role in the lectin pathway. The MBL-associated serine proteases together with C1r and C1s, the protease subcomponents of the first component of the classical pathway (C1), form a family of enzymes with identical domain organization (7Volanakis J.E. Arlaud G.J. Frank M. Volanakis J.E. The Human Complement System in Health and Disease. Marcel-Dekker, New York1998: 49-81Crossref Google Scholar). The C-terminal trypsin-like serine protease (SP) domain is preceded by five noncatalytic modules. At the N terminus, there is a C1r/C1s/sea urchin Uegf/bone morphogenic protein (CUB) domain followed by an epidermal growth factor (EGF)-like module and a second CUB domain. This N-terminal CUB1-EGF-CUB2 region is responsible for the inter-subunit interactions (e.g. interaction between the proteases and the recognition subunits). The following two complement control protein modules (CCPs), which associate directly with the SP domain, stabilize the structure of the SP domain and are involved in the proteolytic process. In the case of C1s and MASP-2, which share almost the same substrate specificity, the CCPs were shown to provide accessory binding sites for the C4 substrate and thereby increase catalytic efficiency (8Rossi V. Bally I. Thielens N.M. Esser A.F. Arlaud G.J. J. Biol. Chem. 1998; 273: 1232-1239Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar). The CCPs, however, do not increase the efficiency of C2 cleavage, indicating that the two substrates bind to different regions of the enzymes. MASP-1, MASP-2, and C1r are capable of autoactivation, where the zymogen proteases become cleaved and activated without the contribution of any extrinsic cleavage factor. The autoactivation is an inherent property of the serine protease domains, the other modules are not involved in this process (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar, 10Kardos J. Gál P. Szilágyi L. Thielens N.M. Szilágyi K. Lőrincz Z. Kulcsár P. Gráf L. Arlaud G.J. Závodszky P. J. Immunol. 2001; 167: 5202-5208Crossref PubMed Scopus (36) Google Scholar). Our main priority was to characterize the structural background of the autoactivation process. Recently, the three-dimensional structure of the activated MASP-2 CCP2-SP fragment has been solved (11Harmat V. Gál P. Kardos J. Szilágyi K. Ambrus G. Végh B. Náray-Szabó G. Závodszky P. J. Mol. Biol. 2004; 342: 1533-1546Crossref PubMed Scopus (69) Google Scholar). The structure revealed the background of some important physiological properties of the MASP-2 enzyme. In this report, we characterize the zymogen catalytic fragment (CCP1-CCP2-SP) of MASP-2 and describe its x-ray structure. Based upon the zymogen and active structures, we present models for the autoactivating complex and propose a mechanism for autoactivation. Mutagenesis, Expression, and Purification of MASP-2 CCP1-CCP2-SP R444Q Mutant—Mutagenesis was performed with the QuikChange ® site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Recombinant plasmid for expression of wild type MASP-2 CCP1-CCP2-SP was used as template. Recombinant protein expression and renaturation were performed as described earlier (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar). The renatured protein solution was concentrated on ultrafiltration membrane (Millipore Corp., Bedford, MA), and it was carried through its isoelectric point (pI 5.6) by dropping it into a 0.5 m sodium acetate buffer (pH 5.0). The solution was dialyzed against 50 mm NaOAc, 0.5 mm EDTA (pH 5.0) and filtered on a 0.45-μm nitrocellulose membrane. The renaturated protein was purified on a Mono S HR 5/5 column (Amersham Biosciences). It was eluted with a linear NaCl gradient from 200 to 600 mm. The collected fraction was once again carried through the isoelectric point by dropping it into a 1 m HEPES buffer (pH 7.4), and it was dialyzed against 20 mm HEPES, 145 mm NaCl, 5 mm EDTA (pH 7.4). The purification steps were monitored on SDS-PAGE. Purification of C4—Human C4 was prepared from 20 ml of fresh serum according to the methods of Dodds (12Dodds A.W. Methods Enzymol. 1993; 223: 46-61Crossref PubMed Scopus (90) Google Scholar). The obtained protein was ∼70% pure, and it was dialyzed against 20 mm HEPES, 145 mm NaCl, 0.5 mm EDTA (pH 7.4). Small C4 aliquots were frozen in liquid nitrogen and kept at -80 °C. They were thawed only once and used up within 3 days. Purification of Wild Type Zymogen MASP-2 CCP1-CCP2-SP—The expression, solubilization, renaturation, and dialysis were performed according to Ref. 9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar. The protein was purified at the same conditions as it was in the case of the R444Q mutant except that the entire procedure was carried out at 4 °C. Zymogen aliquots (1.93 μm) were frozen in liquid nitrogen and kept at -20 °C. Right before usage, zymogen aliquots, 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. The concentration of zymogen MASP-2 CCP1-CCP2-SP was 1.93μm. 12-14 samples were taken at various time points within 60 min from the beginning of incubation. An estimated half-life was given for the zymogen by measuring the diminution of the CCP1-CCP2-SP chain and the appearance of the SP domain on reducing SDS-PAGE. The quantification of these data was made by using a GEL DOC 1000 instrument and Molecular Analyst software for densitometric calculations (Bio-Rad). 2-4 parallel experiments were analyzed to determine the half-life of wild type zymogen. Activation of MASP-2 CCP1-CCP2-SP R444Q Mutant—The R444Q (2.95 μm) mutant was activated by thermolysin as described in Ref. 13Lacroix M. Ebel C. Kardos J. Dobó J. Gál P. Závodszky P. Arlaud G.J. Thielens N.M. J. Biol. Chem. 2001; 276: 36233-36240Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar. C4 Cleavage—To measure the kinetic parameters of the C4 cleavage by MASP-2 CCP1-CCP2-SP R444Q mutant and by the thermolysin-activated mutant, they were incubated with C4 at 37 °C. Serial dilutions were made from mutant and substrate to find the optimal, well characterizable conditions. The concentration was 2.85 × 10-8 m, 6.11 × 10-9 m, and 6.08 × 10-7 m for the uncleaved R444Q mutant, the thermolysin-activated R444Q mutant, and the C4 substrate, respectively. Typically, 11-13 samples were taken within 55 min from the beginning of the reaction at various times. Data from 2-4 independent measurements were used for the calculations. The kinetic parameters were determined by visualizing and measuring the diminution of the α chain of C4 on Coomassie-stained SDS-PAGE using a GEL DOC 1000 instrument and m, and 6.08 × 10 Molecular analyst software (Bio-Rad) for densitometric calculations. The reactions were assumed to be of the Michaelis-Menten type. The kinetic constants kcat, Km, and kcat/Km were estimated by unbiased, non-linear regression methods, regressing the data on the following equation: t = ([S0] - [S] + Km × ln([S0]/[S]))/(kcat× [E0]). In the presence of C1 inhibitor, no cleavage could be detected, so this reaction was used as a 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 μm) was incubated with a 10-fold excess of R444Q (0.64 μm) mutant. Serial dilutions of the wild type zymogen were made to find a concentration at which the rate of autoactivation was slow enough. The incubations were carried out in 20 mm HEPES, 145 mm NaCl, 0.5 mm EDTA (pH 7.4) buffer at 37 °C for 3 h. All of the samples were visualized on SDS-PAGE and were analyzed by densitometry as described above. The appearance of the SP domain was followed. Cleavage of Synthetic Substrate—The cleavage rates of MASP-2 CCP1-CCP2-SP fragments on the synthetic substrate benzyloxycarbonyl-Gly-Arg-S-benzyl (MP Biomedicals Inc., Aurora, OH) were obtained as described in Ref. 11Harmat V. Gál P. Kardos J. Szilágyi K. Ambrus G. Végh B. Náray-Szabó G. Závodszky P. J. Mol. Biol. 2004; 342: 1533-1546Crossref PubMed Scopus (69) Google Scholar. Differential Scanning Calorimetry—Calorimetric measurements were performed on a VP-DSC (MicroCal) differential scanning calorimeter. Denaturation curves were recorded between 20 and 80 °C at a pressure of 2,5 atm, using a scanning rate of 1 °C/min. The protein concentration was set to 0.2 mg/ml. Samples were dialyzed against 20 mm Hepes (pH 7.4), 145 mm NaCl, and the dialysis buffer was used as a reference. Heat capacities were calculated as outlined by Privalov (14Privalov P.L. Adv. Protein Chem. 1979; 33: 167-241Crossref PubMed Scopus (2203) Google Scholar). Crystallographic studies on zymogen MASP-2 R444Q Mutant—Crystals of the zymogen MASP-2 CCP1-CCP2-SP R444Q mutant fragment were grown using the hanging drop method at 20 °C. Crystals were obtained by mixing 2 μl of reservoir solution and 2 μl of protein solution. The reservoir solution contained 20% polyethylene glycol 6000, 0.2 m NaCl, 10% glycerol, and 0.1 m Tris-HCl, pH 8.0. The protein solution contained 1 mg/ml MASP-2 fragment in a buffer of 20 mm Tris/HCl, pH 7.4, and 0.03% NaN3. Data were collected using the ID14 EH4 beam line at the European Synchrotron Radiation Facility at cryogenic temperatures. Data were processed with the XDS program package; they were scaled, merged, and reduced with XSCALE (15Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). The structure was solved by molecular replacement using the program MOLREP (16Vagin A.A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar) of Collaborative Computing Project 4 (17Collaborative Computational Project 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). The SP domain of the MASP-2 activated structure (11Harmat V. Gál P. Kardos J. Szilágyi K. Ambrus G. Végh B. Náray-Szabó G. Závodszky P. J. Mol. Biol. 2004; 342: 1533-1546Crossref PubMed Scopus (69) Google Scholar) (Protein Data Bank accession code 1Q3X) was used as a search model. Refinement was carried out with the REFMAC5 program (18Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar), using restrained maximum likelihood refinement and TLS refinement (19Winn M. Isupov M. Murshudov G.N. Acta Crystallogr. Sect. D. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar). ARP (20Lamzin V.S. Wilson K.S. Methods Enzymol. 1997; 277: 269-305Crossref PubMed Scopus (279) Google Scholar) was used for automatic solvent building. Model building was carried out using the O program (21Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). The final model contains protein residues 296-686 with the exception of residue 661. The stereochemistry of the structure was assessed with PROCHECK (22Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Data collection and refinement statistics are shown in TABLE ONE.TABLE ONEData collection and refinement statistics of the zymogen R444Q mutant form of MASP-2 CCP1-CCP2-SP fragmentCrystal parametersSpace groupP212121Cell constantsa = 47.665 Å, b = 72.689 Å, c = 110.989 ÅData qualityResolution range (last resolution shell)28.868–2.18Å (2.25–2.18 Å)RmeasaRmeas = (Σh(n/(n – 1))0.5Σj|〈I〉h – Ihj|)/(ΣhjIhj) with 〈Ih〉 = (ΣjIhj)/nj0.099 (0.575)Completeness88.1% (43.1%)No. of observed/unique151,887/18,330 (1508/800)I/σ(I)15.13 (1.90)Refinement residualsR0.207Rfreeb5.1% of the reflections in a test set for monitoring the refinement process0.253Model qualityRoot 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 residues390Protein atoms/water molecules2910/84Residues in dual conformations1Residues with disordered side chains23Disordered residues1a Rmeas = (Σh(n/(n – 1))0.5Σj|〈I〉h – Ihj|)/(ΣhjIhj) with 〈Ih〉 = (ΣjIhj)/njb 5.1% of the reflections in a test set for monitoring the refinement process Open table in a new tab Molecular Modeling of the Enzyme-Substrate Complex of MASP-2—We docked the P4-P3′ (Thr441-Gly447) segment of zymogen MASP-2 to the substrate binding site of the active structure. The AutoDock 3.05 (23Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9209) Google Scholar) program has been used for docking the zymogen MASP-2 heptapeptide fragment to the active form. The charges were assigned to the structures by using SYBYL 6.5 (Tripos Associates Inc., St. Louis, MO). The initial conformation of the fragment was built and optimized by SYBYL 6.5. AutoDockTools was used to define the rotable bonds of the fragment. We used a Lamarckian genetic algorithm for the docking with local search, parameterized according to previous systematic optimization studies (24Thomsen R. BioSystems. 2003; 72: 57-73Crossref PubMed Scopus (54) Google Scholar) (ga_run = 200; generation = 27.000; ga_num_evals = 25.000.000; ga_pop_size = 100, number of active torsion = 18). In order to fit the zymogen MASP-2 to the docked fragment, we mutated back the structure in silico according to the wild type (Q444R). To have a realistic model, we built a conformational library of the flexible loop of zymogen MASP-2 that contains our heptapeptide fragment (Arg439-Gly448) by Swiss PDB viewer (25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9589) Google Scholar). A selection of energetically favorable loop conformations has been root mean square deviation-fitted to the docked heptapeptide. The Gly442-Tyr446 peptide fragment of zymogen MASP-2 was replaced by the corresponding docked fragment. The obtained complex structure of zymogen and activated MASP-2 was energy-minimized, relaxed at 310 K by molecular dynamics simulation in water using GROMACS (26Lindahl E. Hess B. van der Spoel D. J. Mol. Mod. 2001; 7: 306-317Crossref Google Scholar). Coordinates of the MASP-2-MASP-2 complex model are available upon request. Design of Stable Zymogen MASP-2 CCP1-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 possesses inherent autoactivating capacity and requires no extrinsic enzymatic factors for the autoactivation to occur. Wild type MASP-2, similarly to other trypsin-like proteases, can be activated through the cleavage of an Arg-Ile bond at the N-terminal region of the catalytic SP domain. We designed, constructed, and expressed an R444Q mutant of the catalytic fragment of MASP-2, thereby ensuring that the protein does not undergo autoactivation during the enzymatic characterization, crystallization, and structure determination processes. Gln was chosen on the basis of isomorphic replacement (27Tüdős E. Cserző M. Simon I. Int. J. Pept. Protein Res. 1990; 36: 236-239Crossref PubMed Scopus (36) Google Scholar) and was predicted to have the least probability to interfere with the protein fold. Stability of Zymogen MASP-2 CCP1-CCP2-SP R444Q Mutant—We expressed the R444Q mutant of the catalytic CCP1-CCP2-SP fragment of MASP-2 in E. coli cells. The wild type activated form of this fragment of MASP-2 has already been successfully expressed in the same expression system, and its biochemical properties have been characterized (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar). After renaturation, the R444Q mutant was purified to homogeneity by ion exchange chromatography. The purified protein migrated as a single band of 44 kDa on the reducing SDS-PAGE (Fig. 1), indicating that no cleavage occurred within the polypeptide chain during expression and purification. In contrast to the R444Q mutant, the wild type fragment was fully activated after the same treatment. To assess the stability of the purified R444Q mutant, it was labeled with 125I and was incubated with either buffer or human plasma at 37 °C for 24 h. The samples were run on SDS-PAGE, and the proteins were visualized by autoradiography. No cleavage product could be observed (data not shown) demonstrating the stability of the R444Q zymogen even upon prolonged incubation at physiological conditions. Folding of Zymogen MASP-2 CCP1-CCP2-SP R444Q Mutant—To further characterize the folding and stability of our stable zymogen R444Q mutant, we measured the melting profile of the active wild type and the zymogen R444Q mutant by differential scanning calorimetry. As the melting curves demonstrate (Fig. 2), both fragments show a sharp, cooperative melting transition, indicating a compact, folded structure. The melting point of the active fragment (50.8 °C) is 2.6 °C higher than that of the zymogen form (48.2 °C), and the calorimetric enthalpy change is also larger in the case of the active species. This difference can be explained by the stabilization effect of the activation process. During activation, the loosely bound, flexible loops of the activation domain become part of the more compact activated structure. The zymogen mutant MASP-2 fragment showed no detectable activity on synthetic substrate (benzyloxycarbonyl-Gly-Arg-S-benzyl) even at very high levels of enzyme concentration (∼200 μm), indicating that the catalytic machinery is disrupted in the zymogen and there is no active trypsin-like serine protease contamination in the purified material. To confirm that the zymogen mutant MASP-2 CCP1-CCP2-SP fragment is correctly folded and can be converted into an active enzyme, it was treated with thermolysin (a non-trypsin-like) protease to specifically cleave the Gln444-Ile445 bond (13Lacroix M. Ebel C. Kardos J. Dobó J. Gál P. Závodszky P. Arlaud G.J. Thielens N.M. J. Biol. Chem. 2001; 276: 36233-36240Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The R444Q mutant was activated using limited proteolysis by thermolysin (Fig. 1), giving rise to an active MASP-2 species with activity on a synthetic and a protein substrate comparable with that of the wild type MASP-2 fragment (TABLE TWO). The recovery of enzymatic activity of the R444Q mutant following thermolysin cleavage underlined that it is suitable for studying the structural and functional properties of zymogen MASP-2.TABLE TWOCleavage rates of MASP-2 CCP1-CCP2-SP and its R444Q mutant on benzyloxycarbonyl-Gly-Arg-S-benzyl(Z-Gly-Arg-S-Bzl) synthetic substrate and on C4 protein substrateEnzymeZ-Gly-Arg-S-Bzl kcat/KmC4 kcat/Kmm–1 s–1MASP-2 CCP1-CCP2-SP9.40 × 105 ± 6.2 × 1045.50 × 105 ± 5×104aData from Ambrus et al. (9)MASP-2 CCP1-CCP2-SP R444Q activated by thermolysin1.30 × 105 ± 1.7 × 1038.50 × 105 ± 2.5×105MASP-2 CCP1-CCP2-SP R444Q–bBelow the detection limit of the assay7.36 × 104 ± 1.0×104a Data from Ambrus et al. (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar)b Below the detection limit of the assay Open table in a new tab 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 protein substrate of wild-type MASP-2 (29Chen C.-B. Wallis R. J. Biol. Chem. 2004; 279: 26058-26065Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This is most probably due to the accessory C4 binding sites on the CCP2 module of MASP-2 (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar). We incubated our stable zymogen R444Q MASP-2 with human C4 at 37 °C. To our surprise, zymogen MASP-2 was able to cleave C4 with a high efficiency (Fig. 3, TABLE THREE). This activity was completely abolished in the presence of C1 inhibitor, indicating that it was mediated by zymogen MASP-2 and not by other (potentially contaminating) protease. The fact that the Km values for the zymogen and the active enzyme are in the same range indicates that the accessory C4 binding site is present on both forms of the enzyme, and it is not affected by the conformation change of MASP-2 activation. Since zymogen MASP-2 showed no activity on synthetic substrate but was shown to cleave C4, we argue that the one-chain zymogen form of MASP-2 can adopt an active-like conformation, and this conformational change may be induced by the large protein substrate C4. Previously, it was shown that trypsinogen can be converted into an active state upon strong ligand binding (e.g. pancreatic trypsin inhibitor and Ile-Val dipeptide) without proteolytic cleavage (30Bode W. Schwanger P. Huber R. J. Mol. Biol. 1978; 118: 99-112Crossref PubMed Scopus (245) Google Scholar). A physiologically important example of a proteolytically active serine protease zymogen is tissue-type plasminogen activator, which has significant (10-20%) activity relative to the two-chain form (31Renatus M. Engh R.A. Stubbs M.T. Huber R. Fischer S. Kohnert U. Bode W. EMBO J. 1997; 16: 4797-4805Crossref PubMed Scopus (93) Google Scholar). The proteolytic activity of zymogen MASP-2 could be responsible for the first step of the autoactivation process, where a zymogen MASP-2 molecule cleaves and activates another zymogen MASP-2 molecule.TABLE THREEKinetic parameters of MASP-2 CCP1-CCP2-SP and its R444Q mutant on C4 substrateEnzymekcatKmkcat/Kms–1mm–1 s–1MASP-2 CCP1-CCP2-SP0.90 ± 0.41.60 × 10–6 ± 5 × 10–65.50 × 105 ± 5 × 104aData from Ambrus et al. (9)MASP-2 CCP1-CCP2-SP R444Q0.026 ± 0.0063.77 × 10–7 ± 1.5 × 10–77.36 × 104 ± 1.0 × 104a Data from Ambrus et al. (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar) Open table in a new tab Autoactivation of MASP-2—The observed rate of MASP-2 autoactivation is concentration-dependent. At low concentrations (∼0.1 μm) during renaturation, the catalytic fragment of wild type MASP-2 remains zymogen for several days. However, rapid autoactivation occurs during the subsequent concentration and purification steps (9Ambrus G. Gál P. Kojima M. Szilágyi K. Balczer J. Antal J. Gráf L. Laich A. Moffat B.E. Schwaeble W. Sim R.B. Závodszky P. J. Immunol. 2003; 170: 1374-1382Crossref PubMed Scopus (175) Google Scholar). To prepare wild type zymogen MASP-2, we adjusted the pH to 5.0 immediately after renaturation and performed the subsequent chromatographic steps at 4 °C. At pH 5.0, the histidine residue in the catalytic triad becomes protonated, and the rate of the proteolysis decreases dramatically. Using this purification strategy, we managed to prepare wild type zymogen MASP-2, which remains relatively stable at pH 5.0 even at a relatively high concentration (3 μm). At 4 °C, it autoactivates slowly (its half-life is approximately 2 weeks), but at 37 °C and pH 7.5, its half-life is only 26 min. The activation curve of the wild type zymogen MASP-2 shows the typical features of a true autoactivation process; it has a sigmoid shape with a lag phase at the beginning (Fig. 4). In the lag phase of the autoactivation process the first reaction step dominates where zymogen molecules cleave zymogen molecules. It" @default.
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• W2097771036 title "A True Autoactivating Enzyme" @default.
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