Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.

Mannose (or mannan)-binding lectin (MBL),3 a member of the collectins (1), is a Ca2+-dependent serum lectin that recognizes carbohydrates such as mannose and N-acetylglucosamine (GlcNAc) (2) on the surfaces of pathogens and plays a role in innate immunity through activating complement (3, 4, 5). MBL is an oligomer of subunits composed of three identical polypeptide chains comprising a cysteine-rich, a collagen-like, a neck, and a carbohydrate recognition domain. MBL forms a complex with C1r/C1s-like serine proteases termed MASPs (MBL-associated serine proteases), and activates complement via the lectin pathway (6, 7). To date, two types of MASP, MASP-1 (8, 9) and MASP-2 (10, 11), have been identified in human MBL preparations. MASP-1, MASP-2, C1r, and C1s constitute a subfamily of the serine protease family (12). Their structure shares six domains, a first CUB domain, an epidermal growth factor-like domain, a second CUB domain, two complement control protein domains, and a serine protease domain (13, 14). Recently, a protein designated sMAP (small MBL-associated protein) (15) or MAp19 (MBL-associated plasma protein of 19 kDa) (16) has been identified. sMAP is identical with MASP-2 through the first two domains followed by four unique amino acids at the C-terminal, which are derived from a specific exon of the MASP-2 gene. The physiological role of sMAP remains unknown. In blood, MBL forms complexes (MBL-complexes) with the proenzyme forms of MASP-1 and MASP-2 and with sMAP. Upon binding of MBL to its ligands, MASPs are activated by a split in the polypeptide chain, generating two polypeptides linked by a disulfide bond, thus acquiring proteolytic activities. It has recently been demonstrated that a serum GlcNAc-binding lectin termed ficolin/P35 with collagen-like and fibrinogen-like domains is also associated with MASPs and sMAP (17).

MASP-1 was first discovered and characterized as a serine protease capable of activating C4, C2 (8), and C3 (18). In a subsequent report, Thiel et al. (10) separated human MASP-1 and MASP-2 by SDS-PAGE and demonstrated that when blotted onto a membrane MASP-2 activates C4, whereas MASP-1 does not.

C1 inhibitor (C1 INH) is a plasma protein that belongs to the serpin superfamily of serine protease inhibitors, and is in blood involved in the regulation of proteolytic systems such as the coagulation, fibrinolytic, and complement systems (19). In the complement system, C1 INH exhibits inhibitory activities against C1r and C1s, thus playing a role in the regulation of classical pathway activation. The importance of C1 INH is revealed by genetically determined C1 INH deficiency that causes hereditary angioedema (HAE).

The present study describes the separation of MASP-1 and MASP-2 and their activities against complement components. The effects of C1 INH on the proteolytic activities of MASP-1 and MASP-2 were also examined to elucidate the regulation mechanism of the lectin pathway.

Mannan from Saccharomyces cerevisiae was purchased from Sigma (St. Louis, MO). (Amidinophenyl)methanesulfonyl fluoride (p-APMSF) and mannose were from Wako Pure Chemical Industries (Osaka, Japan). p-Nitrophenyl-p-guanidinobenzoate (NPGB) was from Merck (Rahway, NJ). CNBr-activated Sepharose 4B was from Amersham Pharmacia (Uppsala, Sweden). Mouse mAbs against MBL (3E7) (20) and MASP-1 (1E2) (21) and rabbit polyclonal Abs against a synthetic peptide representing 19 C-terminal amino acids of MASP-1 (10) were prepared as described previously. Anti-MASP-2 Ab was from rabbits immunized with a synthetic peptide corresponding to the first 20 N-terminal amino acid residues of MASP-2 that was conjugated to a multiple Ag peptide backbone (22). Coupling of mannan, anti-MBL (3E7), or anti-MASP-1 (1E2) to CNBr-activated Sepharose 4B was performed according to the manufacturer’s instructions. Human C3, C4 (23), C2 (24), oxidized C2 (25), and C1 INH (26) were prepared as previously described. Veronal-buffered saline (VB) is a 10-mM solution of Veronal containing 0.148 M NaCl (pH 7.4). EDTA-GVB is VB supplemented with 10 mM EDTA and 0.1% gelatin. MGVB is a 5-mM solution of Veronal containing 0.074 M NaCl, 0.1% gelatin, 2.3% mannitol, 2 mM CaCl2, and 0.5 mM MgCl2.

MASP-1 and MASP-2 in proenzyme forms were isolated from human serum as described previously (9, 15, 27). In brief, human serum was passed through a yeast mannan-Sepharose column using a 10 mM imidazole buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl2, 0.2 mM NPGB, 20 μM p-APMSF, and 2% mannitol. Proenzymes MASP-1 and MASP-2 complexed with MBL were eluted with the above buffer containing 0.3 M mannose. To separate proenzymes MASP-1 and MASP-2 from MBL, preparations containing the complex were applied to anti-MBL-Sepharose and then MASPs were eluted with imidazole buffer containing 20 mM EDTA and 1 M NaCl. Finally, proenzymes MASP-1 and MASP-2 were separated by passing through anti-MASP-1-Sepharose in the same buffer as used for the anti-MBL-Sepharose. MASP-2 was recovered in the effluents, whereas MASP-1 was eluted with 0.1 M glycine buffer (pH 2.2).

Human MBL-complexes, in which MASP-1 and MASP-2 were in activated forms, were isolated from serum. For this, human serum was first applied to a mannan-Sepharose column equilibrated with 50 mM Tris buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl2, 0.2 mM NPGB, and 20 μM p-APMSF. After washing with starting buffer without NPGB and p-APMSF, elution was conducted with the same buffer containing 0.3 M mannose. The MBL-complex eluate was next applied to the anti-MBL-Sepharose column equilibrated with the same buffer. MBL-complexes were eluted from the column with glycine buffer. After dialysis against 50 mM Tris buffer containing 1 M NaCl, 20 mM EDTA, the MBL-complex preparation was applied to an anti-MBL-Sepharose column. The effluent contained a mixture of MASP-1, MASP-2, and sMAP, whereas MBL was retained and subsequently eluted with glycine buffer. The preparation containing MASP-1, MASP-2, and sMAP was applied to an anti-MASP-1-Sepharose column equilibrated with the same buffer as used for the second anti-MBL-Sepharose. At this step, MASP-2 passed through, whereas MASP-1 was retained on the column and eluted with glycine buffer. sMAP was found in both the MASP-1 and the MASP-2 fractions. The fractions containing MASP-1 or MASP-2 were pooled and used to study the effects of C1 INH on MASP activity.

SDS-PAGE was performed according to the Laemmli method. After transferring proteins from the gels to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blots were probed with anti-MASP-1 peptide or anti-MASP-2 peptide Abs. Peroxidase-conjugated anti-rabbit IgG was used as a second Ab, and the blot was developed with a Konica Immunostain kit (Konica, Tokyo, Japan).

C4 consumption was assayed as described previously (8). In brief, 50 μl of sample containing MASP-1 or MASP-2 diluted in MGVB was incubated with 50 μl of C4 (two site-forming units, SFU) at 37°C for 30 min. The reaction mixtures were further incubated for 60 min with 100 μl of 50-fold-diluted C4-deficient guinea pig serum and 100 μl of sheep erythrocytes (108/ml) bearing anti-sheep erythrocytes Abs (EA). The lytic reaction was terminated by the addition of 1 ml of EDTA-GVB. After centrifugation, the OD of the supernatant was determined at 414 nm. The hemolytic rate (y) and the average number of hemolytic sites per cell (z) defined as z = −ln(1 − y) was calculated. The percentage consumption was determined by the following formula: % consumption = (Z1 − Z2)/Z1 × 100, where Z1 = z value in the absence of sample and Z2 = z value in the presence of sample.

EA bearing human C4b (EAC4b) was prepared as described previously (23). Fifty microliters of samples, 50 μl of oxidized human C2 (2 SFU), and 100 μl of EAC4b (108/ml) were incubated in MGVB at 30°C for 10 min, and then 200 μl of 50-fold-diluted guinea pig serum with EDTA-GVB (C-EDTA) was added to the reaction mixture as a source of C3 to C9. After additional incubation at 37°C for 60 min, 1 ml of EDTA-GVB was added to terminate the reaction. From the OD determined at 414 nm, z was calculated as described above.

C3 activation was assayed as described previously (18). In brief, 10 μl of samples and 10 μl of human C3 (2 μg) in VB was incubated at 37°C for 60 min, and the reaction mixture was subjected to SDS-PAGE (7.5% gel) under reducing conditions.

MASP-1 or MASP-2 was incubated with C1 INH at 37°C for 30 min. The mixtures were then subjected to SDS-PAGE under nonreducing conditions followed by immunoblotting.

Five microliters of fractions containing MASP-2 in MGVB were incubated with 45 μl of various amounts of C1 INH diluted in MGVB at 37°C for 15 min and then with 50 μl of C4 at 37°C for 30 min. Residual hemolytic activity of C4 was assayed as described above (C4 consumption), and the effect of C1 INH on MASP-2-mediated C4 activation was expressed as the percentage inhibition by the following formula: % inhibition = (Z3 − Z2)/(Z1 −Z2) × 100, where Z1 = z value in the absence of MASP-2 and C1 INH, Z2 = z value in the presence of MASP, and Z3 = z value in the presence of MASP and C1INH. For inhibition of C2 activation, 25 μl of MASP-1 or MASP-2 were incubated with 25 μl of various amounts of C1 INH, 50 μl of oxidized C2, and 100 μl of EAC4b at 30°C for 10 min and then with C-EDTA at 37°C for 1 h, and the effect of C1 INH on MASP-1 and on MASP-2 was expressed as the percentage inhibition by the following formula: % inhibition = Z2/(Z1 − Z2) × 100, where Z1 = z value in the absence of MASP-2 and Z2 = z value in the presence of MASP-2.

For direct observation of the effect of C1 INH on MASP-1, 10 μl of MASP-1 were incubated with 10 μl of various amounts of C1 INH at 37°C for 30 min and then with 10 μl of C3 (2 μg) for 60 min. The reaction mixtures were analyzed by SDS-PAGE (7.5% gel) under reducing conditions.

To obtain human MASP-1 and MASP-2 in proenzyme forms, the MBL-complex was first prepared from serum using a mannan column in the presence of serine protease inhibitors. This preparation also contained IgG and IgM. Further purification was achieved using an anti-MBL column. The MBL-complex was bound to the anti-MBL column, and MASPs and sMAP were then eluted with EDTA at a high salt concentration, whereas MBL was retained on the column. Finally, the eluate containing MASPs and sMAP was subjected to affinity chromatography on an anti-MASP-1 column. At this step, MASP-2 passed through the column, whereas MASP-1 was retained on the column and could subsequently be eluted with an acidic buffer (Fig. 1). Most of sMAP coeluted with MASP-1. MASP-1 and MASP-2 obtained in this way showed a single band under reducing conditions with molecular size of ∼93 kDa and 70 kDa, respectively, indicating that both MASPs were in proenzyme forms.

FIGURE 1.

Separation of the proenzyme forms of MASP-1 and MASP-2 on an anti-MASP-1 column. The MBL-complexes were purified on mannan-Sepharose and MBL was separated from MASPs and sMAP on anti-MBL-Sepharose. MASP-1 and MASP-2 in proenzyme forms and sMAP were then subjected to affinity chromatography on anti-MASP-1 (1E2)-Sepharose. The effluent (fractions 2–10) and eluate (fractions 12, 13, 14, and 16) were subjected to SDS-PAGE (12% gel) under reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250 (A). After SDS-PAGE, immunoblotting was performed using anti-MASP-1 (B) or anti-MASP-2 (C). Arrow in B indicates proenzyme MASP-1. Upper and lower arrows in C indicate proenzyme MASP-2 and sMAP, respectively. Right ordinates, Molecular mass markers.

FIGURE 1.

Separation of the proenzyme forms of MASP-1 and MASP-2 on an anti-MASP-1 column. The MBL-complexes were purified on mannan-Sepharose and MBL was separated from MASPs and sMAP on anti-MBL-Sepharose. MASP-1 and MASP-2 in proenzyme forms and sMAP were then subjected to affinity chromatography on anti-MASP-1 (1E2)-Sepharose. The effluent (fractions 2–10) and eluate (fractions 12, 13, 14, and 16) were subjected to SDS-PAGE (12% gel) under reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250 (A). After SDS-PAGE, immunoblotting was performed using anti-MASP-1 (B) or anti-MASP-2 (C). Arrow in B indicates proenzyme MASP-1. Upper and lower arrows in C indicate proenzyme MASP-2 and sMAP, respectively. Right ordinates, Molecular mass markers.

Close modal

Based on the results that proenzymes MASP-1 and MASP-2 were separated on the anti-MASP-1 column, we next isolated MASP-1 and MASP-2 in activated forms. The MBL-complex was prepared utilizing mannan and anti-MBL columns. After dialysis against a buffer containing EDTA and 1 M NaCl, the MBL-complex from the anti-MBL column was applied to the anti-MBL column again. In this buffer, MASP-1, MASP-2, and sMAP passed through the column and thus separated from MBL. This preparation was finally fractionated on an anti-MASP-1 column. MASP-2 passed through the column, whereas MASP-1 was retained on the column and could be eluted with an acidic buffer as was the case with the MASP proenzymes (Fig. 2). Both MASP-1 and MASP-2 were in activated forms as revealed by the presence of the L chain of MASP-1 and the H chain of MASP-2. sMAP was found to copurify with both MASP-1 and MASP-2.

FIGURE 2.

Separation of MASP-1 and MASP-2 in activated forms on an anti-MASP-1 column. A preparation of MASPs and sMAP was obtained as the effluent from anti-MBL-Sepharose and applied to an anti-MASP-1 (1E2)-Sepharose column. The effluent (fractions 1–10) and eluate (fractions 12–20) were analyzed for the presence of the components by SDS-PAGE (12% gel) under reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250 (A). After SDS-PAGE, immunoblotting was performed using Abs against MASP-1 peptide (B) or MASP-2 peptide (C). Arrow in B indicates the L chain of MASP-1. Upper and lower arrows in C indicate the H chain of MASP-2 and sMAP, respectively. Right ordinates, Molecular mass markers.

FIGURE 2.

Separation of MASP-1 and MASP-2 in activated forms on an anti-MASP-1 column. A preparation of MASPs and sMAP was obtained as the effluent from anti-MBL-Sepharose and applied to an anti-MASP-1 (1E2)-Sepharose column. The effluent (fractions 1–10) and eluate (fractions 12–20) were analyzed for the presence of the components by SDS-PAGE (12% gel) under reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250 (A). After SDS-PAGE, immunoblotting was performed using Abs against MASP-1 peptide (B) or MASP-2 peptide (C). Arrow in B indicates the L chain of MASP-1. Upper and lower arrows in C indicate the H chain of MASP-2 and sMAP, respectively. Right ordinates, Molecular mass markers.

Close modal

Preparations containing either MASP-1 or MASP-2 in activated forms were tested for proteolytic activities against C4, C2, and C3. C4 consumption and C2 activation by MASPs were determined hemolytically as described in Materials and Methods. C3 cleavage by MASP was directly assessed by SDS-PAGE. As shown in Fig. 3, C4 consumption was observed with the fractions containing MASP-2 but not with those containing MASP-1. Both MASP-1 and MASP-2 activated C2. C3 cleavage with an appearance of the α-chain was noted for MASP-1 but not for MASP-2. In contrast with the activated forms of MASPs, proenzymes MASP-1 and MASP-2 showed no proteolytic activities against C4, C2, and C3 (data not shown), indicating that at the conditions of the experiment no activation of the proenzymes occurred.

FIGURE 3.

Proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3. The effluent fractions from anti-MASP-1-Sepharose containing MASP-2 (fractions 1–4) and the eluate containing MASP-1 (fractions 12–16) from anti-MASP-1-Sepharose were analyzed for the proteolytic activities. Top (□), C4 consumption. Samples of each fraction were incubated with C4 and residual hemolytic activities of C4 were determined. Middle (▪), C2 activation. Samples of each fraction were incubated with EAC4b and C2 and then with C3 to C9 (guinea pig serum diluted with EDTA-GVB). From hemolytic rates, z was determined as described in Materials and Methods. Bottom (SDS-PAGE), C3 cleavage. Samples of each fraction or buffer (left lane) were incubated with C3 and the reaction mixtures were subjected to SDS-PAGE (7.5% gel) under reducing conditions. The gel was stained with Coomassie Brilliant Blue R-250.

FIGURE 3.

Proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3. The effluent fractions from anti-MASP-1-Sepharose containing MASP-2 (fractions 1–4) and the eluate containing MASP-1 (fractions 12–16) from anti-MASP-1-Sepharose were analyzed for the proteolytic activities. Top (□), C4 consumption. Samples of each fraction were incubated with C4 and residual hemolytic activities of C4 were determined. Middle (▪), C2 activation. Samples of each fraction were incubated with EAC4b and C2 and then with C3 to C9 (guinea pig serum diluted with EDTA-GVB). From hemolytic rates, z was determined as described in Materials and Methods. Bottom (SDS-PAGE), C3 cleavage. Samples of each fraction or buffer (left lane) were incubated with C3 and the reaction mixtures were subjected to SDS-PAGE (7.5% gel) under reducing conditions. The gel was stained with Coomassie Brilliant Blue R-250.

Close modal

C1 INH forms stable complexes with C1s and C1r in a 1:1 ratio and inhibits their proteolytic activities. To determine the effect of C1 INH on MASP-1 or MASP-2, we first tested for covalent complex formation between C1 INH and MASPs in activated forms. C1 INH was incubated with MASP-1 or MASP-2 in activated forms at 37°C for 1 h and then subjected to SDS-PAGE followed by immunoblotting. As shown in Fig. 4,A, a novel band with an apparent m.w. of 196 kDa reacting with anti-MASP-1 Ab appeared after incubation of C1 INH with MASP-1. The molecular size of this band almost matched the sum of MASP-1 (81 kDa) and C1 INH (98 kDa). Similarly, incubation of MASP-2 (63 kDa) with C1 INH resulted in an appearance of a new band (175 kDa) (Fig. 4 B). These results indicate that C1 INH formed equimolar complexes with MASP-1 and MASP-2.

FIGURE 4.

Complex formation between MASPs and C1 INH. A, MASP-1 was incubated with buffer (lanea) or with C1 INH (laneb). As a control, C1 INH alone (lane c) was incubated. The reaction mixtures were analyzed by SDS-PAGE (6% gel) under nonreducing conditions, followed by immunoblotting using anti-MASP-1. Upper arrow and lower arrow indicate C1 INH-MASP-1 and MASP-1, respectively. B, MASP-2 was incubated with buffer (laned) or C1 INH (lanee). Lanef is C1 INH alone, and analyzed by SDS-PAGE (6% gel) followed by immunoblotting using anti-MASP-2. Upper arrow and lower arrow indicate C1 INH-MASP-2 and MASP-2, respectively. Ordinate, Molecular mass markers.

FIGURE 4.

Complex formation between MASPs and C1 INH. A, MASP-1 was incubated with buffer (lanea) or with C1 INH (laneb). As a control, C1 INH alone (lane c) was incubated. The reaction mixtures were analyzed by SDS-PAGE (6% gel) under nonreducing conditions, followed by immunoblotting using anti-MASP-1. Upper arrow and lower arrow indicate C1 INH-MASP-1 and MASP-1, respectively. B, MASP-2 was incubated with buffer (laned) or C1 INH (lanee). Lanef is C1 INH alone, and analyzed by SDS-PAGE (6% gel) followed by immunoblotting using anti-MASP-2. Upper arrow and lower arrow indicate C1 INH-MASP-2 and MASP-2, respectively. Ordinate, Molecular mass markers.

Close modal

We next determined whether C1 INH inhibits the proteolytic activities of MASP-1 and MASP-2 in activated forms. The proteolytic activities of MASP-1 against C3 and C2 were examined in the presence of various amounts of C1 INH. As shown in Fig. 5,A, C3 cleavage by MASP-1 was inhibited by C1 INH in a dose-dependent manner. Similarly, C1 INH inhibited C2 activation mediated by MASP-1 (Fig. 5,B). Fig. 5, C and D, depict the results of the effects of C1 INH on MASP-2 activities against C4 and C2. Both activities of MASP-2 were inhibited by C1 INH in a dose-dependent manner.

FIGURE 5.

Inhibition of proteolytic activities of MASPs by C1 INH. A, The effect of C1 INH on the proteolytsis of C3 by MASP-1. A fixed mixture of C3 and MASP-1 was incubated with various amounts of C1 INH before SDS-PAGE. The C1 INH concentrations were: lanea, 0 μg/ml; laneb, 20 μg/ml; lanec, 10 μg/ml; laned, 5 μg/ml; lanee was C3 alone. B and D, The effect of C1 INH on the proteolysis of C2 by MASP-1 (B) or MASP-2 (D). Under the conditions in which both MASP-1 and MASP-2 showed the z value of 1 in the absence of C1 INH, MASP-1 or MASP-2 was incubated with EAC4b, C2 and various amounts of C1 INH and then with C3 to C9. From hemolytic rates, percent inhibition was determined as described in Materials and Methods. Results are mean ± SD (n = 3). C, The effect of C1 INH on the proteolysis of C4 by MASP-2. Under the conditions in which MASP-2 showed 60% consumption of C4 in the absence of C1 INH, MASP-2 was incubated with various amounts of C1 INH and then with C4. From residual hemolytic activity of C4, percent inhibition was determined as described in Materials and Methods. Results are mean ± SD (n = 3).

FIGURE 5.

Inhibition of proteolytic activities of MASPs by C1 INH. A, The effect of C1 INH on the proteolytsis of C3 by MASP-1. A fixed mixture of C3 and MASP-1 was incubated with various amounts of C1 INH before SDS-PAGE. The C1 INH concentrations were: lanea, 0 μg/ml; laneb, 20 μg/ml; lanec, 10 μg/ml; laned, 5 μg/ml; lanee was C3 alone. B and D, The effect of C1 INH on the proteolysis of C2 by MASP-1 (B) or MASP-2 (D). Under the conditions in which both MASP-1 and MASP-2 showed the z value of 1 in the absence of C1 INH, MASP-1 or MASP-2 was incubated with EAC4b, C2 and various amounts of C1 INH and then with C3 to C9. From hemolytic rates, percent inhibition was determined as described in Materials and Methods. Results are mean ± SD (n = 3). C, The effect of C1 INH on the proteolysis of C4 by MASP-2. Under the conditions in which MASP-2 showed 60% consumption of C4 in the absence of C1 INH, MASP-2 was incubated with various amounts of C1 INH and then with C4. From residual hemolytic activity of C4, percent inhibition was determined as described in Materials and Methods. Results are mean ± SD (n = 3).

Close modal

The binding between C1q, C1r, and C1s in the C1 complex is facilitated by Ca2+, and the complex is formed in a 1:2:2 stoichiometry. On the other hand, the mode of binding and stoichiometry of the complex composed of MBL, MASP-1, MASP-2, and sMAP remains unsolved. In the present report, we showed that MASP-1, MASP-2, and sMAP dissociate from MBL in the presence of EDTA and high concentration of salt (1 M NaCl). Several lines of evidence have revealed that EDTA alone is insufficient, and both EDTA and high concentration of salt are required for the dissociation of MBL from the other components, suggesting that the complex formation is facilitated by a combination of Ca2+ and presumably electrostatic interactions (28, 29). When the mixture of MASP-1, MASP-2, and sMAP from the anti-MBL column was applied to an anti-MASP-1 column with buffer containing EDTA and 1 M NaCl, MASP-2 was recovered in the pass-through fractions, whereas MASP-1 was retained on the column and could be eluted with an acidic buffer. Two explanations can be proposed for the separation of MASP-1 and MASP-2 on this column; MASP-1 and MASP-2 form a complex in a Ca2+-dependent manner or, alternatively, MASP-1 and MASP-2 are independently complexed with MBL.

As shown in Figs. 1 and 2, sMAP copurified on the anti-MASP-1 column with both MASP-1 and MASP-2 when they were in activated forms, whereas most of the sMAP coeluted with MASP-1 when the MASPs were in proenzyme forms. The reason for this difference in the behavior of sMAP remains unclear. One possibility is that a long exposure of MASP-1-sMAP to EDTA and 1 M NaCl during dialysis in the purification step of activated MASPs allowed some dissociation of MASP-1 and sMAP. Alternatively, it could be suggested that sMAP has a lower affinity for activated MASP-1 than for unactivated MASP-1.

Isolated MASP-1 and MASP-2 in activated forms exhibited proteolytic activities against C3 and C4, respectively. The specificity of MASP-2 for C4 is consistent with a previous report (10). Both MASPs activated C2. In this respect, the function of MASP-2 resembles C1s in the C1 complex, whereas MASP-1 shows unique proteolytic activities. Analysis of the cDNA of MASP and C1r/C1s serine protease domains from human, mouse, hamster, Xenopus, carp, shark, lamprey, and ascidian revealed that the MASP/C1r/C1s family falls into two groups (30, 31). The first group, termed “TCN type,” where the serine residue in the active center is encoded by TCN, encompasses human MASP-1, mouse MASP-1, Xenopus MASP-1, ascidian MASPa, and MASPb. The second group, termed “AGY type,” where the serine residue in the active center is encoded by AGY, encompasses human MASP-2, human C1r/C1s, hamster C1s, mouse MASP-2, Xenopus MASP-2, carp MASP, shark MASP, and lamprey MASP. The TCN type possesses a so called “histidine loop” structure, whereas the AGY type does not. It is speculated that the AGY type diverged from the TCN type in the evolution of the MASP/C1r/C1s family (30, 32). The ascidians appear to lack the classical pathway C4 and C2, and the function of ascidian MASPs may thus be restricted to cleavage of C3 (33). This type of substrate selectivity has been preserved in human MASP-1, which, like ascidian MASPs, possesses the histidine loop. The C4 cleaving activity of human MASP-2 and C1s could be speculated to be related to the cutout of the histidine loop. The split exon nature of ascidian MASPs and MASP-1 (30), and the TCN codon contrasting to MASP-2 and C1r/C1s are features of no structural consequence to the proteins, but most useful when trying to sort out the phylogeny. Although the stoichiometry of the MBL-complex and activation mechanism remain unsolved, MASP-2 in the complex is likely to possess the same function as C1s, which cleaves C4 and C2 resulting in the formation of C3 convertase, C4b2a. On the other hand, MASP-1 directly cleaves C3 into C3a and C3b, the latter of which initiates the alternative complement pathway (18) and also acts as an opsonin. The physiological significance of the observed C2 activating-capacity of MASP-1 is unclear. It is also unknown which MASP is more active in cleaving C2 on a molar basis, since the presence of sMAP in both MASP-1 and MASP-2 preparations does not allow quantitative analysis of MASPs. However, it should be noted that more C1 INH was required for preventing proteolysis of C2 by MASP-1 than by MASP-2, suggesting that the proteolytic activity of MASP-1 against C2 might be lower than that of MASP-2.

Unlike C1r or C1s, MASP-1 forms a complex with α2-macroglobulin (α2M) that is a protease inhibitor in blood (34). The effect of α2M on MASP-2 is to be elucidated. In this report, we demonstrated that C1 INH inhibited the proteolytic activities of both MASP-1 and MASP-2 by forming stable complexes in a 1:1 stoichiometry as do C1r and C1s, indicating a function in regulation of lectin pathway activation. Wong et al. (35) also observed that a mixture of activated MASP-1 and MASP-2 interacts with C1 INH, resulting in the formation of complexes between C1 INH and each MASP. In the C1 complex in blood, C1 INH associates noncovalently with proenzyme C1r to prevent its autoactivation. Upon binding of C1 to immune complexes, C1 INH dissociates from proenzyme C1r, resulting in the autoactivation of C1r and the subsequent activation of C1s. Thus, C1 INH also modulates C1 activation by inhibiting the autoactivation of C1r. If MASP-1 and/or MASP-2 autoactivate, it is possible that their autoactivation is also regulated by C1 INH in a manner similar to C1r. The present and previous studies (34, 36) suggest that C1 INH regulates both the classical and lectin pathways and α2M regulates the latter.

The MBL-complex and the C1 complex appear to be similar in that the serine proteases involved in each complex have specific proteolytic activities. However, several features are different between the MBL-complex and C1. First, the MBL-complex possesses sMAP, which has no equivalent in C1, although its role in the complex remains unsolved. Second, as discussed above, it is possible that MASP-1 and MASP-2 are independently associated with MBL.

We thank Akiko Nozawa for technical assistance.

2

Address correspondence to Dr. Misao Matsushita, Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan. E-mail address: mmatsu@fmu.ac.jp

3

Abbreviations used in this paper: MBL, mannose (or mannan)-binding lectin; GVB, gelatin-containing Veronal-buffered saline; VB, Veronal-buffered saline; EDTA-GVB, gelatin-Veronal buffer containing EDTA; C-EDTA, guinea pig serum diluted with EDTA-GVB; C1 INH, C1 inhibitor; CUB, C1r/C1s/Uegf/bone morphogenetic protein 1; EA, sheep erythrocytes sensitized with Ab; EAC4b, EA bearing guinea pig C1 and human C4b; sMAP, small MBL-associated protein; MAp19, MBL-associated plasma protein of 19 kDa; MASP, MBL-associated serine protease; MBL-complex, a complex consisting of MBL, MASP-1, MASP-2, and sMAP; MGVB, gelatin-Veronal buffer containing mannitol, CaCl2, and MgCl2; NPGB, p-nitrophenyl-p-guanidinobenzoate; p-APMSF, (amidinophenyl)methanesulfonyl fluoride.

1
Epstein, J., Q. Eichbaum, S. Sheriff, R. A. B. Ezekowitz.
1996
. The collectins in innate immunity.
Curr. Opin. Immunol.
8
:
29
2
Turner, M. W..
1996
. Mannose-binding lectin: the pluripotent molecule of the innate immune system.
Immunol. Today
17
:
532
3
Ikeda, K., T. Sannoh, N. Kawasaki, T. Kawasaki, I. Yamashina.
1987
. Serum lectin with known structure activates complement through the classical pathway.
J. Biol. Chem.
262
:
7451
4
Ohta, M., M. Okada, I. Yamashina, T. Kawasaki.
1990
. The mechanism of carbohydrate-mediated complement activation by the serum mannan-binding protein.
J. Biol. Chem.
265
:
1980
5
Lu, J., S. Thiel, H. Wiedemann, R. Timpl, K. B. M. Reid.
1990
. Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q.
J. Immunol.
144
:
2287
6
Matsushita, M..
1996
. The lectin pathway of the complement system.
Microbiol. Imuunol.
40
:
887
7
Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, Jr, R. A. B. Ezekowitz.
1999
. Phylogenetic perspectives in innate immunity.
Science
284
:
1313
8
Matsushita, M., T. Fujita.
1992
. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.
J. Exp. Med.
176
:
1497
9
Matsushita, M., Y. Endo, T. Fujita.
1998
. MASP1.
Immunobiology
199
:
340
10
Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen, K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. Holmskov, K. B. M. Reid, J. C. Jensenius.
1997
. A second serine protease associated with mannan-binding lectin that activates complement.
Nature
386
:
506
11
Vorup-Jensen, T., J. C. Jensenius, S. Thiel.
1998
. MASP-2, the C3 convertase generating protease of the MBLectin complement activating pathway.
Immunobiology
199
:
348
12
Matsushita, M., Y. Endo, M. Nonaka, T. Fujita.
1998
. Complement-related serine proteases in tunicates and vertebrates.
Curr. Opin. Immunol.
10
:
29
13
Sato, T., Y. Endo, M. Matsushita, T. Fujita.
1994
. Molecular characterization of a novel serine protease involved in activation of the complement system by mannose-binding protein.
Int. Immunol.
6
:
665
14
Takada, F., H. Takayama, H. Hatsuse, M. Kawakami.
1994
. A new member of the C1s family of component protein found in a bactericidal factor, Ra-reactive factor, in human serum.
Biochem. Biophys. Res. Commun.
196
:
1003
15
Takahashi, M., Y. Endo, T. Fujita, M. Matsushita.
1999
. A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway.
Int. Immunol.
11
:
859
16
Stover, C. D., S. Thiel, M. Thelen, N. J. Lynch, T. Vorup-Jensen, J. C. Jensenius, W. J. Schwaeble.
1999
. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of the complement are encoded by a single structural gene.
J. Immunol.
162
:
3481
17
Matsushita, M. Y. Endo, T. Fujita.
2000
. Complement-activating complex of ficolin and mannose-binding lectin-associated serine protease.
J. Immunol.
164
:
2281
18
Matsushita, M., T. Fujita.
1995
. Cleavage of the third component of complement (C3) by mannose-binding protein-associated serine protease (MASP) with subsequent complement activation.
Immunobiology
194
:
443
19
Davis, A. E., III.
1997
. C1 inhibitor: functional analysis of naturally-occuring mutant proteins.
Adv. Exp. Med. Biol.
425
:
185
20
Matsushita, M., Takahashi A, H. Hatsuse, M. Kawakami, T. Fujita.
1992
. Human mannose-binding protein is identical to a component of Ra-reactive factor.
Biochem. Biophys. Res. Commun.
183
:
645
21
Terai, I., K. Kobayashi, M. Matsushita, T. Fujita.
1997
. Human serum mannose-binding lectin-associated serine protease-1 (MASP-1): determination of levels in body fluids and identification of two forms in serum.
Clin. Exp. Immunol.
110
:
317
22
Posnett, D. N., J. P. Tam.
1989
. Multiple antigenic peptide method for producing antipeptide site-specific antibodies.
Methods Enzymol.
178
:
739
23
Matsushita, M., H. Okada.
1986
. Alternative complement pathway activation by C4b deposited during classical pathway activation.
J. Immunol.
136
:
2994
24
Nagasawa, S., R. M. Stroud.
1977
. Cleavage of C2 by C1s into the antigenicaly distinct fragments C2a and C2b: demonstration of binding of C2b to C4b.
Proc. Natl. Acad. Sci. USA
74
:
2998
25
Polly, M. J., H. J. Müller-Eberhard.
1967
. Enhancement of the hemolytic activity of the second component of complement by oxidation.
J. Exp. Med.
126
:
1013
26
Sim, R. B., A. Reboul.
1981
. Preparation and properties of human C1 inhibitor.
Methods Enzymol.
80
:
43
27
Matsushita, M., R. A. B. Ezekowitz, T. Fujita.
1995
. The Gly-54 Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.
Biochem. J.
311
:
1021
28
Tan, S. M., M. C. M. Chung, O. L. Kon, S. Thiel, S. H. Lee, J. Lu.
1996
. Improvements on the purification of mannan-binding lectin and demonstration of its Ca2+-independent association with a C1s-like serine protease.
Biochem. J.
319
:
329
29
Thiel, S., S. Petersen, T. Vorup-Jensen, M. Matsushita, T. Fujita, C. Stover, W. Schwaeble, J. Jensenius.
2000
. Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2 and the MBL associated protein, MAp19.
J. Immunol.
165
:
878
30
Endo, Y., M. Takahashi, M. Nakao, H. Saiga, H. Sekine, M. Matsushita, M. Nonaka, T. Fujita.
1998
. Two lineages of mannose-binding lectin-associated serine protease (MASP) in vertebrates.
J. Immunol.
161
:
4924
31
Ji, X., K. Azumi, M. Sasaki, M. Nonaka.
1997
. Ancient origin of the complement lectin pathway revealed by molecular cloning of mannan-binding protein-serine protease from a urochorate, the Japanese ascidian, Halocynthia roretzi. Proc.
Natl. Acad. Sci. USA
94
:
6340
32
Endo, Y., T. Sato, M. Matsushita, T. Fujita.
1996
. Exon structure of the gene encoding the human mannose-binding protein-associated serine protease (MASP) light chain: Comparison with complement C1r and C1s genes.
Int. Immunol.
8
:
1355
33
Nonaka, M., K. Azumi, X. Ji, C. N. Yanaka, M. Sasaki, H. Saiga, A. W. Dodds, H. Sekine, M. K. Homma, M. Matsushita, Y. Endo, T. Fujita.
1999
. Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi.
J. Immunol.
162
:
387
34
Terai, I., K. Kobayashi, M. Matsushita, T. Fujita, K. Matsuno.
1995
. α2-macroglobulin binds to and inhibits mannose-binding protein-associated serine protease.
Int. Immunol.
7
:
1579
35
Wong, N. K. H., M. Kojima, J. Dobo, G. Ambrus, R. B. Sim.
1999
. Activities of the MBL-associated serine proteases (MASPs) and their regulation by natural inhibitors.
Mol. Immunol.
36
:
853
36
Sim, R. B., A. Reboul, G. J. Arlaud, C. L. Villers, M. G. Colomb.
1979
. Interaction of 125I-labelled complement subcomponents C1r and C1s with protease inhibitors in plasma.
FEBS Lett.
97
:
111