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• W2005402525 abstract "Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation. Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation. mannan-binding lectin MBL-associated serine protease serine protease domain p-tosyl-l-arginine methyl ester N-carboxybenzyloxyglycine-l-arginine thiobenzyl ester N-acetylglycine-l-lysine methyl ester Nα-benzoyl-l-arginine ethyl ester complement control protein module protein module originally found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1 diisopropyl fluorophosphate polyacrylamide gel electrophoresis α2-macroglobulin; the nomenclature of protein modules is that defined by Bork and Bairoch (1Bork P. Bairoch A. Trends Biochem. Sci. 1995; 20 (suppl.): C03Google Scholar) Mannan-binding lectin (MBL)1 is an oligomeric C-type lectin that recognizes arrays of neutral carbohydrates such as mannose and N-acetylglucosamine on the surface of pathogenic microorganisms (2Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (852) Google Scholar). This selectivity endows MBL with the ability to discriminate self from infectious non-self, and confers this “ante-antibody” a major role in innate immunity, as underlined by numerous clinical reports indicating that MBL deficiency is linked with increased susceptibility to infectious diseases (3Turner M.W. Immunol. Today. 1996; 17: 532-540Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 4Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2153) Google Scholar, 5Turner M.W. Hamvas R.M.J. Rev. Immunogenetics. 2000; 2: 305-322PubMed Google Scholar). In addition to its role as an opsonin (3Turner M.W. Immunol. Today. 1996; 17: 532-540Abstract Full Text PDF PubMed Scopus (678) Google Scholar), MBL has devised the ability to associate to several modular proteases termed MASPs (MBL-associated serine proteases) (6Thiel 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, 7Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (560) Google Scholar, 8Takada F. Takayama Y. Hatsuse H. Kawakami M. Biochem. Biophys. Res. Commun. 1993; 196: 1003-1009Crossref PubMed Scopus (60) Google Scholar, 9Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar). A single MASP entity was initially identified, and characterized as a protease with the ability to cleave complement proteins C4, C2, and C3 (7Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (560) Google Scholar, 10Matsushita M. Fujita T. Immunobiology. 1995; 194: 443-448Crossref PubMed Scopus (127) Google Scholar). Further studies by Thiel et al. (6Thiel 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) revealed that MASP was indeed a mixture of two related but distinct proteases, MASP-1 and MASP-2, and that only the latter had the ability to cleave C4. A third protein component MAp19, arising from alternative splicing of the MASP-2 gene (11Stover C.M. Thiel S. Thelen M. Lynch N.J. Vorup-Jensen T. Jensenius J.C. Schwaeble J.W. J. Immunol. 1999; 162: 3481-3490PubMed Google Scholar, 12Takahashi M. Endo Y. Fujita T. Matsushita M. Int. Immunol. 1999; 11: 859-864Crossref PubMed Scopus (170) Google Scholar), and very recently a further protease MASP-3 (13Dahl M.R. Thiel S. Willis A.C. Vorup-Jensen T. Christensen T. Petersen S.V. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) were also shown to be associated with MBL. MASP-1 and MASP-2 show a domain organization identical to that of C1r and C1s, the enzymatic components of the C1 complex of complement (14Arlaud G.J. Volanakis J.E. Thielens N.M. Narayana S.V.L. Rossi V. Xu Y. Adv. Immunol. 1998; 69: 249-307Crossref PubMed Google Scholar), with an N-terminal CUB module (15Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (521) Google Scholar) followed by an epidermal growth factor-like module, a second CUB module, two contiguous CCP modules (16Reid K.B.M. Bentley D.R. Campbell R.D. Chung L.P. Sim R.B. Kristensen T. Tack B.F. Immunol. Today. 1986; 7: 230-234Abstract Full Text PDF PubMed Scopus (187) Google Scholar), and a C-terminal chymotrypsin-like serine protease domain (see Fig. 1). By analogy with human C1s, it may be anticipated that the proteolytic activity and specificity of the MASPs is defined by the two CCP modules together with the serine protease domain (17Rossi 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), and that the latter forms a rigid association with the preceding, second CCP module (18Gaboriaud C. Rossi V. Bally I. Arlaud G.J. Fontecilla-Camps J.C. EMBO J. 2000; 19: 1755-1765Crossref PubMed Scopus (93) Google Scholar). Comparative analysis of the cDNAs of C1r, C1s, and the MASPs in different animal species reveal that these fall into two groups (19Ji X. Azumi K. Sasaki M. Nonaka M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6340-6345Crossref PubMed Scopus (145) Google Scholar,20Endo Y. Takahashi M. Nakao M. Saiga H. Sekine H. Matsushita M. Nonaka M. Fujita T. J. Immunol. 1998; 161: 4924-4930PubMed Google Scholar). In the smaller and probably more ancient group comprising MASP-1 and the ascidian MASPs, the active site serine is encoded by a “TCN” codon (where N is A, T, G, or C), and the histidine-loop disulfide bridge is present. In the larger group encompassing C1r, C1s, MASP-2, and most of the known animal MASPs, the active site serine is encoded by an “AGY” codon (where Y is T or C), and the histidine-loop is missing. The only available information dealing with the substrate specificity of MASP-1 and MASP-2 has been obtained on proteases isolated from human serum, and is somewhat controversial. Thus, whereas it is now widely accepted that MASP-2 cleaves both C4 and C2 (6Thiel 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, 21Matsushita M. Thiel S. Jensenius J.C. Terai I. Fujita T. J. Immunol. 2000; 165: 2637-2642Crossref PubMed Scopus (264) Google Scholar, 22Wong N.K.H. Kojima M. Dobo J. Ambrus G. Sim R.B. Mol. Immunol. 1999; 36: 853-861Crossref PubMed Scopus (64) Google Scholar), the observation that MASP-1 can cleave C3 and hence directly trigger complement activation (21Matsushita M. Thiel S. Jensenius J.C. Terai I. Fujita T. J. Immunol. 2000; 165: 2637-2642Crossref PubMed Scopus (264) Google Scholar, 23Matsushita M. Endo Y. Fujita T. Immunobiol. 1998; 199: 340-347Crossref PubMed Scopus (45) Google Scholar) is debated (22Wong N.K.H. Kojima M. Dobo J. Ambrus G. Sim R.B. Mol. Immunol. 1999; 36: 853-861Crossref PubMed Scopus (64) Google Scholar, 24Zhang Y. Suankatray C. Jones D.R. Zhang X.H. Lint T.F. Gewurz H. Mol. Immunol. 1998; 35 (abstr.): 390Crossref Google Scholar). In addition, the relative efficiency of MASP-2 with respect to C4 and C2 cleavage has not been assessed. The objective of the present work was to produce recombinant human MASP-1 and MASP-2 and catalytic fragments thereof, to precisely determine their substrate specificity on both protein substrates and synthetic esters, and to measure the kinetic parameters of their activity. Our data reveal that MASP-2 cleaves C4 much more efficiently than does C1s, emphasizing the physiological relevance of MASP-2 with respect to complement activation. In contrast, recombinant MASP-1 shows only marginal C3 cleaving activity, raising questions about its involvement in complement activation. Diisopropyl phosphorofluoridate was from Acros Organics, Noisy le Grand, France. The plasmids containing the full-length MASP-1 and MASP-2 cDNAs were obtained as described previously (6Thiel 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, 25Vorup-Jensen T. Ph. D. Thesis. University of Aarhus, 2000Google Scholar). Oligonucleotides were obtained from Oligoexpress, Paris, France. Trypsin was obtained from Sigma, Saint Quentin Fallavier, France. Z-Gly-Arg-S-Bzl was from Enzyme Systems Products, Livermore, CA. Ac-Gly-Lys-OMe, Bz-Arg-OEt, and Tos-Arg-OMe were obtained from Sigma. MBL was isolated from human plasma according to the procedure described by Tan et al. (26Tan S.M. Chung M.C.M. Kon O.L. Thiel S. Lee S.H. Lu J. Biochem. J. 1996; 319: 329-332Crossref PubMed Scopus (95) Google Scholar), with a further purification step using ion-exchange chromatography, as described in Thielens et al. (27Thielens N.M. Cseh S. Thiel S. Vorup-Jensen T. Rossi V. Jensenius J.C. Arlaud G.J. J. Immunol. 2001; 166: 5068-5077Crossref PubMed Scopus (114) Google Scholar). Recombinant full-length MASP-1 was expressed using a baculovirus insect cell system and purified by ion-exchange chromatography and affinity chromatography on an UltraLinkTM-MBL column, as described previously (27Thielens N.M. Cseh S. Thiel S. Vorup-Jensen T. Rossi V. Jensenius J.C. Arlaud G.J. J. Immunol. 2001; 166: 5068-5077Crossref PubMed Scopus (114) Google Scholar). Activated C1s and complement proteins C4, C2, and C3 were purified from human plasma according to published procedures (28Arlaud G.J. Reboul A. Sim R.B. Colomb M.G. Biochim. Biophys. Acta. 1979; 576: 151-162Crossref PubMed Scopus (47) Google Scholar, 29Dodds A.W. Methods Enzymol. 1993; 223: 46-61Crossref PubMed Scopus (90) Google Scholar, 30Thielens N.M. Villiers M.B. Reboul A. Villiers C.L. Colomb M.G. FEBS Lett. 1982; 141: 19-24Crossref PubMed Scopus (17) Google Scholar, 31Al Salihi A. Ripoche J. Pruvost L. Fontaine M. FEBS Lett. 1982; 150: 238-242Crossref Scopus (28) Google Scholar). Partially purified C5 was prepared as described by Al Salihi et al.(31Al Salihi A. Ripoche J. Pruvost L. Fontaine M. FEBS Lett. 1982; 150: 238-242Crossref Scopus (28) Google Scholar), and partially purified factor B was obtained from the flow-through fraction of the last C2 purification step on C4b-Sepharose (30Thielens N.M. Villiers M.B. Reboul A. Villiers C.L. Colomb M.G. FEBS Lett. 1982; 141: 19-24Crossref PubMed Scopus (17) Google Scholar). C1 inhibitor was purified from human plasma essentially as described in Ref. 32Reboul A. Arlaud G.J. Sim R.B. Colomb M.G. FEBS Lett. 1977; 79: 45-50Crossref PubMed Scopus (55) Google Scholar, except that concanavalin A-agarose (Sigma) was used instead of concanavalin A-Sepharose. Purified human α2-macroglobulin (α2M) was kindly provided by L. Sottrup-Jensen (University of Aarhus). The concentrations of purified proteins were determined using the following absorption coefficients ( A 1cm1% at 280 nm) and molecular weights: C1s, 14.5 and 79,800; C4, 8.3 and 205,000; C2, 8.9 and 102,000; C3, 10.0 and 185,000 (29Dodds A.W. Methods Enzymol. 1993; 223: 46-61Crossref PubMed Scopus (90) Google Scholar, 30Thielens N.M. Villiers M.B. Reboul A. Villiers C.L. Colomb M.G. FEBS Lett. 1982; 141: 19-24Crossref PubMed Scopus (17) Google Scholar, 33Pétillot Y. Thibault P. Thielens N.M. Rossi V. Lacroix M. Coddeville B. Spik G. Schumaker V.N. Gagnon J. Arlaud G.J. FEBS Lett. 1995; 358: 323-328Crossref PubMed Scopus (25) Google Scholar). The absorption coefficients ( A 1cm1% at 280 nm) used for the recombinant fragments CCP1/2-SP and CCP2-SP of MASP-2 (18.3 and 19.2, respectively) were calculated from the number of Trp, Tyr, and disulfides according to the method of Edelhoch (34Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar), using molecular weight values of 42,710 and 35,330, calculated from the sequence (6Thiel 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). Because of the low amounts of material recovered, the concentrations of full-length MASP-1 and MASP-2 and of the CCP1/2-SP fragment of MASP-1 were estimated on the basis of Coomassie Blue staining after SDS-PAGE analysis using appropriate internal standards and respective molecular weights of 82,000, 74,200, and 45,200 (6Thiel 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,9Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar). A DNA fragment encoding residues 299–699 of human MASP-1 was amplified by polymerase chain reaction using the VentR polymerase and the MASP-1b/pDR2ΔEF1α vector (25Vorup-Jensen T. Ph. D. Thesis. University of Aarhus, 2000Google Scholar) as a template, according to established procedures. The sequence of the sense primer (5′-GAAGATCTCAATGAGTGCCCAGAGCT-3′) introduced aBglII restriction site (underlined) and allowed in-frame cloning with the melittin signal peptide of the pNT-Bac expression vector (17Rossi 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). The antisense primer (5′-GGAATTCTCAGTTCCTCACTCC-3′) introduced a stop codon (bold face) followed by an EcoRI site (underlined) at the 3′ end of the fragment. The polymerase chain reaction product was digested with BglII and EcoRI, purified, and cloned into the BamHI/EcoRI sites of the pNT-Bac vector. The DNA fragment encoding the MASP-2 signal peptide plus the mature protein (amino acids 1–671) was excised from the pBS-MASP-2 vector (6Thiel 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) by digestion with XhoI andEcoRI and cloned into the corresponding restriction sites of the pFastBac1 baculovirus transfer vector (Life Technologies, Inc.). DNA fragments encoding residues 298–686 (CCP1/2-SP construct) and residues 365–686 (CCP2-SP construct) of human MASP-2 were amplified by polymerase chain reaction using the VentR polymerase and the pFastBac MASP-2 vector as a template. The sequences of the sense primers (5′-CGGGATCCCCAGCCTTGCCCTTATCC-3′ for the CCP1/2-SP construct and 5′-CGGGATCCCGACTGTGGCCCTCCTG-3′ for the CCP2-SP construct) introduced a BamHI restriction site (underlined) and allowed in-frame cloning with melittin signal peptide of the pNT-Bac expression vector. The antisense primer (5′-GGAATTCTTAAAAATCACTAATTATGTTCTC-3′) was identical for both constructs and introduced a stop codon (bold face) followed by an EcoRI site (underlined). Both polymerase chain reaction fragments were digested with EcoRI and BamHI, purified, and cloned into the corresponding sites of the pNT-Bac vector. All DNA constructs used in this study were checked for the absence of mutations by double-stranded DNA sequencing (Genome Express, Grenoble). The insect cells Spodoptera frugiperda (Ready-Plaque Sf9 cells from Novagen) andTrichoplusia ni (High FiveTM) were routinely grown and maintained as described previously (27Thielens N.M. Cseh S. Thiel S. Vorup-Jensen T. Rossi V. Jensenius J.C. Arlaud G.J. J. Immunol. 2001; 166: 5068-5077Crossref PubMed Scopus (114) Google Scholar). Recombinant baculoviruses were generated using the Bac-to-BacTM system (Life Technologies, Inc.). The bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen S.A., Courtaboeuf, France) and used to transfect Sf9 insect cells utilizing cellfectin in Sf900 II SFM medium (Life Technologies, Inc.) as described in the manufacturer's protocol. Recombinant virus particles were collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee (35King L.A. Possee R.D. The Baculovirus Expression System: A Laboratory Guide. Chapman and Hall Ltd., London1992: 111-114Google Scholar). High Five cells (1.75 × 107 cells/175-cm2 tissue culture flask) were infected with the recombinant viruses at a multiplicity of infection of 2–3 in Sf900 II SFM medium at 28 °C for 48 h (MASP-2 fragments) or 72 h (full-length MASP-2 and CCP1/2-SP fragment of MASP-1). Culture supernatants were collected by centrifugation. The supernatant containing MASP-2 was dialyzed against 50 mm NaCl, 1 mm EDTA, 50 mmtriethanolamine hydrochloride, pH 8.5, and loaded onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 × 10 cm) equilibrated in the same buffer. Elution was carried out by applying a 800-ml linear gradient from 50 to 500 mm NaCl in the same buffer. Fractions containing the recombinant protein were identified by Western blot analysis, and dialyzed against 50 mm NaCl, 1 mm EDTA, 50 mm triethanolamine hydrochloride, pH 8.0. Further purification was achieved by ion-exchange chromatography on a Mono-Q HR 5/5 column (Amersham Pharmacia Biotech) equilibrated in the same buffer. Elution was carried out with a linear NaCl gradient from 50 to 500 mm in 90 min. The supernatants containing the CCP1/2-SP and CCP2-SP fragments of MASP-2 were dialyzed against 5 mm EDTA, 20 mmNa2HPO4, pH 8.6, and loaded onto a Q-Sepharose Fast Flow column (2.8 × 10 cm) equilibrated in the same buffer. Elution was carried out by applying a 700-ml linear gradient from 0 to 350 mm NaCl in the same buffer. Fractions containing the recombinant proteins were identified by Western blot analysis, dialyzed against 1.5 m(NH4)2SO4, 0.1 mNa2HPO4, pH 7.4, and further purified by high-pressure hydrophobic interaction chromatography on a TSK-Phenyl 5PW column (Beckman) equilibrated in the same buffer. Elution was carried out by decreasing the (NH4)2SO4 concentration from 1.5m to 0 in 30 min. Both purified recombinant fragments were dialyzed against 145 mm NaCl, 50 mmtriethanolamine hydrochloride, pH 7.4, concentrated up to 0.2 mg/ml by ultrafiltration, and stored at −20 °C. The supernatant containing the CCP1/2-SP fragment of MASP-1 was dialyzed against 50 mm Na acetate, pH 5.1, and loaded on a SP-Sepharose column (Amersham Pharmacia Biotech) (2.8 × 8 cm) equilibrated in the same buffer. Elution was performed with a 800-ml linear gradient from 0 to 600 mm NaCl. Recombinant proteins were dialyzed against 50 mm triethanolamine hydrochloride, 145 mm NaCl, pH 7.4, concentrated by ultrafiltration to 0.05–0.5 mg/ml, and stored at 0 °C. SDS-PAGE analysis was performed as described previously (36Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). Western blot analysis and immunodetection of the recombinant proteins were carried out as described by Rossi et al. (17Rossi 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), or using the ECL detection procedure of Amersham Pharmacia Biotech. The antibodies used were the mouse monoclonal anti-MASP-2 antibody 1.3B7, a rabbit polyclonal anti-MASP-2 antibody (37Petersen S.V. Poulsen K. Stover C.M. Koch C. Vorup-Jensen T. Thiel S. Mol. Immunol. 1998; 35 (abstr.): 409Crossref Google Scholar), and a rabbit anti-peptide antibody directed against the serine protease domain of MASP-1 (6Thiel 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). N-terminal sequence analyses were performed after SDS-PAGE and electrotransfer, using an Applied Biosystems model 477A protein sequencer as described previously (38Rossi V. Gaboriaud C. Lacroix M. Ulrich J. Fontecilla-Camps J.C. Gagnon J. Arlaud G.J. Biochemistry. 1995; 34: 7311-7321Crossref PubMed Scopus (41) Google Scholar). The proteolytic activity of MASP-1, MASP-2, and C1s toward C2, C3, C4, C5, and factor B was measured by incubation at different enzyme:protein ratios for varying periods at 37 °C, as indicated in the text. The extent of reaction was determined after SDS-PAGE analysis under reducing conditions by gel scanning of the cleavage fragments, as described previously (17Rossi 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). For determination of the kinetic parameters, the concentrations of C2 ranged from 1.0 to 4 µm, and those of C4 ranged from 50 to 500 nm (MASP-2 cleavage) or from 0.5 to 4 µm (C1s cleavage). Fixed enzyme concentrations of 2 nm were used in all cases, and enzyme dilutions were performed in 50 mm triethanolamine HCl, 145 mmNaCl, pH 7.4, containing 1 mg/ml ovalbumin. The kinetic constants were determined by the Lineweaver-Burk method using linear regression analysis and are based on duplicate or triplicate measurements of initial rates at 6 separate substrate concentrations. All kinetic analyses were conducted in 150 mm NaCl, 5 mmEDTA, 20 mm sodium phosphate, pH 7.4. Complex formation between C1 inhibitor and MASP-1 or MASP-2 was assessed by incubation of the proteases with excess molar ratios of C1 inhibitor for 15 min at 37 °C in 50 mm triethanolamine-HCl, 145 mmNaCl, pH 7.4, followed by SDS-PAGE analysis under reducing conditions. C1 inhibitor-protease complexes were revealed by Western blot analysis using a rabbit polyclonal antibody directed against full-length MASP-2, and a rabbit anti-peptide antibody directed against the serine protease domain of MASP-1. Titration of the enzymatic activity of C1s, MASP-1, and MASP-2 by C1 inhibitor was performed by preincubating these proteases (at concentrations of 0.25 µm, 20 nm, and 0.25 µm, respectively) with increasing concentrations of C1 inhibitor (10–500 nm) for 15 min at 37 °C. The residual C2 cleaving activity of MASP-2 and C1s was then measured by incubating the enzymes (3.75 and 7.5 nm, respectively) for 15 min at 37 °C in the presence of 2.25 µm C2, followed by SDS-PAGE analysis, as described above. In the case of MASP-1, the residual esterolytic activity was measured on Bz-Arg-OEt, as described above. The reactivity of C1s and MASP-2 toward α2M was measured by preincubating the enzymes (0.25 µm each) for 30 min at 37 °C in the presence of increasing concentrations (3.7–300 µm) of α2M. Residual C4 cleaving activity was then measured by incubating the enzymes (1.25 nm each) for 15 min at 37 °C in the presence of 2.25 µm C4, followed by SDS-PAGE analysis. Esterolytic activities were measured on the synthetic esters Ac-Gly-Lys-OMe and Tos-Arg-OMe using a spectrophotometric assay based on the measurement of methanol released upon hydrolysis (39Arlaud G.J. Thielens N.M. Methods Enzymol. 1993; 223: 61-82Crossref PubMed Scopus (32) Google Scholar). Assays on the thioester Z-Gly-Arg-S-Bzl and on Bz-Arg-OEt were carried out as described by McRae et al.(40McRae B.J. Lin T.-Y,. Powers J.C. J. Biol. Chem. 1981; 256: 12362-12366Abstract Full Text PDF PubMed Google Scholar), and Arlaud and Thielens (39Arlaud G.J. Thielens N.M. Methods Enzymol. 1993; 223: 61-82Crossref PubMed Scopus (32) Google Scholar). All assays were conducted at 30 °C, at substrate concentrations of 0.1–3 mm(Ac-Gly-Lys-OMe and Tos-Arg-OMe), 0.5–3 mm (Bz-Arg-OEt), or 0.075–0.2 mm (Z-Gly-Arg-S-Bzl). The modular structures of MASP-1, MASP-2, and of the truncated fragments used in the present study are depicted in Fig. 1. The recombinant baculoviruses used for expression of each construct were obtained as described under “Experimental Procedures” and used to infect High Five insect cells for various periods at 28 °C. The amount of recombinant material recovered in the culture medium, as estimated by SDS-PAGE and Western blot analysis, ranged from 0.15 (MASP-2) to 2 µg/ml (CCP1/2-SP fragment of MASP-1). Protein purification was performed as described under “Experimental Procedures,” using an initial ion-exchange fractionation step in all cases. Because of the low amounts of material recovered, protein detection and analysis was performed routinely using Western blot analysis rather than Coomassie Blue staining. Due to its very low recovery, recombinant MASP-2 could not be purified to homogeneity since attempts to completely remove contaminant proteins resulted in a nearly complete loss of material. SDS-PAGE analysis of the partially purified MASP-2 fraction under nonreducing conditions revealed two bands reactive with specific antibodies: (i) a major, 80-kDa species corresponding to the full-length protease, yielding two sequences, Thr-Pro-Leu-Gly-Pro-Lys-Trp-Pro-Glu-Pro … and Ile-Tyr-Gly-Gly-Gln-Lys-Ala-Lys-Pro-Gly … , corresponding to the N-terminal ends of the mature protein and of the serine protease domain, respectively; (ii) a 45-kDa species yielding only the first sequence above, and corresponding to a truncated fragment derived from the N-terminal end of the protein. Analysis under reducing conditions indicated that full-length MASP-2 was recovered in a partially activated form, since only 20–30% of the protein migrated as a single chain, proenzyme species, whereas the remainder yielded two bands at 45 and 28 kDa, corresponding to the N-terminal A chain and the serine protease domain, respectively. Based on Coomassie Blue staining after SDS-PAGE analysis, the relative amount of full-length MASP-2 in the partially purified fraction averaged 10% of the total protein contents. Both the CCP1/2-SP and CCP2-SP fragments of MASP-2 could be purified to homogeneity. On SDS-PAGE analysis, the CCP1/2-SP fragment migrated under nonreducing conditions as a 44-kDa band, which upon reduction split into two bands corresponding to the serine protease domain (27 kDa) and the CCP1/2 segment (17 kDa) (Fig.2, lanes 1 and 2), indicating complete activation of the protease. In contrast, the shorter CCP2-SP fragment migrated as a single band of about 35 kDa both under nonreducing and reducing conditions (Fig. 2, lanes 3and 4), indicating that this species had retained a single chain, proenzyme structure. N-terminal sequence analysis of the latter fragment yielded a single sequenceAsp-Pro-Asp-Cys-Gly-Pro-Pro … corresponding to the expected N-terminal end of the fragment, whereas the activated CCP1/2 SP fragment yielded equivalent amounts of two sequences:Asp-Pro-Gln-Pro-Cys-Pro-Tyr … (N-terminal end) and Ile-Tyr-Gly-Gly-Gln-Lys-Ala … (serine protease domain). Protein staining with Coomassie Blue (not shown) yielded the same pictures as observed by Western blot analysis, indicating that no major fragment or contaminant was present in the purified preparations. As in the case of full-length MASP-2, the CCP1/2-SP fragment of MASP-1 could not be purified to homogeneity, mainly because of the low amounts of recombinant material available. SDS-PAGE" @default.
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